Treating and evaluating inflammatory disorders

ABSTRACT

Methods of treating inflammatory disorders, such as rheumatoid arthritis, by modulating TWEAK and TNF-α are disclosed, as are other methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application, filed under 35 U.S.C. § 111, is a continuationclaiming priority under 35 U.S.C. § 120 of International Application No.PCT/US2006/018077, filed on May 10, 2006, which claims priority to U.S.Application Ser. No. 60/679,518, filed on May 10, 2005. The contents ofall the foregoing applications are hereby incorporated by reference.

BACKGROUND

The tumor-necrosis factor (TNF)-related cytokines are a superfamily ofproteins that have an array of functions, including ones implicated inimmune regulation and apoptosis regulation. Examples of TNF superfamilymembers include TNF-α and TWEAK (TNF-like weak inducer of apoptosis).

SUMMARY

As further described below, the TWEAK and TNF-α pathways workindependently to mediate aspects of inflammation. Blocking both of themolecular signalling pathways modulated by TWEAK and TNF-α can be usedto treat a variety of inflammatory disorders. Examples of suchtreatments are described below.

In one aspect, the disclosure features a method of treating a subjectfor an inflammatory disorder. In a preferred embodiment, theinflammatory disorder is an arthritic disorder, e.g., rheumatoidarthritis, psoriatic arthritis, or Sjögren's Syndrome. The methodincludes: administering, to a subject, e.g., a human subject, who has oris at risk for the disorder, e.g., rheumatoid arthritis, a TWEAKblocking agent in combination with a TNF-α blocking agent. The TWEAKblocking agent and the TNF-α blocking agent can be administered inamounts and for a time to provide a therapeutic effect, e.g., an overalltherapeutic effect. The effect can be additive or, in some cases,synergistic. For example, the effect of both blocking agents may be agreater total effect than the sum of the individual effects, e.g., in aparticular subject.

A variety of TWEAK blocking agents can be administered to a subject toblock a interaction or activity of TWEAK or a TWEAK-R. A “TWEAK blockingagent” refers to an agent (e.g., any compound, e.g., an antibody or asoluble form of the TWEAK receptor) that at least partially inhibits aninteraction or activity of a TWEAK or TWEAK-R. For example, the agent atleast partially inhibits an activity, e.g., binding of TWEAK to aTWEAK-R, or the agent at least partially inhibits a nucleic acidencoding TWEAK or TWEAK-R, e.g., to reduce TWEAK or TWEAK-R proteinexpression.

In one embodiment, the agent reduces the ability of TWEAK to bind to aTWEAK receptor, e.g., Fn14. The agent can be a blocking antibody thatbinds to TWEAK or to Fn14. The antibody can be a full length IgG. In oneembodiment, the antibody is human, humanized, or effectively human. Inone embodiment, the TWEAK blocking antibody competes with AB.D3 (anantibody that has ATCC Accession No. HB-12622) for binding with TWEAK,is a humanized antibody AB.D3, comprises at least two, three, four,five, or six CDRs of AB.D3 (or CDRs that are at least overall 85, 90,92, 95, 97% identical to such CDRs), and/or comprises antibody AB.D3variable domains (or one or more variable domains that are at leastoverall 85, 90, 92, 95, 97% identical to such variable domains).

In one embodiment, the agent is a soluble form of a TWEAK receptor,e.g., a human TWEAK receptor such as Fn14. The soluble form of the TWEAKreceptor can be fused to an antibody Fc region (e.g., a human Fcregion). For example, the soluble form of the TWEAK receptor includes asequence at least 95% identical to amino acids 28-X₁ of SEQ ID NO:2,where amino acid X₁ is selected from the group of residues 68 to 80 ofSEQ ID NO:2.

A variety of TNF-α blocking agents can be administered to a subject toblock an interaction or activity of TNF-α or a TNF-α receptor, e.g.,TNFR-I, or TNFR-II. A “TNF-α blocking agent” refers to an agent (e.g.,any compound, e.g., an antibody or a soluble form of a TNF-α receptor)that at least partially inhibits an interaction or activity of TNF-α ora TNF-α receptor. For example, the agent at least partially inhibits anactivity, e.g., binding of TNF-α to a TNF-α receptor, or the agent atleast partially inhibits a nucleic acid encoding TNF-α or a TNF-αreceptor, e.g., to reduce TNF-α or TNF-α receptor protein expression.

In one embodiment, the TNF-α blocking agent reduces the ability of TNF-αto bind to a TNF-α receptor. For example, the TNF-α blocking agentincludes an antibody that binds to TNF-α, TNFR-I, or TNFR-II. Exemplaryantibodies include infliximab or adalimumab. The TNF-α blocking agentcan include a soluble form of a TNF-α receptor and optionally a Fcdomain. For example, the TNF-α blocking agent is etanercept.

As used herein, “administered in combination” means that two or moreagents (e.g., the TWEAK blocking agent and the TNF-α blocking agent) areadministered to a subject at the same time or within an interval, suchthat there is overlap of an effect of each agent on the patient.Preferably the administrations of the first and second agent are spacedsufficiently close together such that a combinatorial effect isachieved. The interval can be an interval of hours, days or weeks.Generally, the agents are concurrently bioavailable, e.g., detectable,in the subject. In a preferred embodiment, at least one administrationof one of the agents, e.g., the first agent (e.g., TNF-α blockingagent), is made while the other agent, e.g., the TWEAK blocking agent,is still present at a therapeutic level in the subject.

In one embodiment, the TWEAK blocking agent is administered between anearlier and a later administration of the TNF-α blocking agent. In otherembodiments, the TNF-α blocking agent is administered between an earlierand a later administration of the TWEAK blocking agent. In a preferredembodiment, at least one administration of one of the agents, e.g., theTNF-α blocking agent, is made within 1, 7, 14, 30, or 60 days of theother agent, e.g., the TWEAK blocking agent.

In one embodiment, prior to administering the TWEAK blocking agent andTNF-α blocking agent, the subject was receiving either the TWEAKblocking agent or TNF-α blocking agent, but not the other. The subjectmay have had a response that did not meet a predetermined threshold,e.g., a stabilization or reduction in a total Sharp score or a Sharperosion score. In another embodiment, the subject can be one who has notbeen previously administered the TNF-α blocking agent nor the TWEAKblocking agent for at least 3 months (e.g., at least 6 months, 9 months,or a year prior) prior to being administered the first and second agentin combination.

In one implementation, the TWEAK blocking agent and TNF-α blocking agentare provided as a co-formulation, and the co-formulation is administeredto the subject. It is further possible, e.g., at least 24 hours beforeor after administering the co-formulation, to administer one of theagents separately from the other. In another implementation, the agentsare provided as separate formulations, and the step of administeringincludes sequentially administering the agents. The sequentialadministrations can be provided on the same day (e.g., within one hourof one another or at least 3, 6, or 12 hours apart) or on differentdays.

Generally, the TWEAK blocking agent and TNF-α blocking agent are eachadministered as a plurality of doses separated in time, e.g., accordingto a regimen. The regimen for one or both may have a regularperiodicity. The regimen for the TNF-α blocking agent can have adifferent periodicity from the regimen for the TWEAK blocking agent,e.g., one can be administered more frequently than the other. The agentscan be administered by any appropriate method, e.g., subcutaneously,intramuscularly, or intravenously. The subject can be administered dosesof the TNF-α blocking agent and doses of the TWEAK blocking agent forgreater than 14 weeks, greater than six or nine months, greater than 1,1.5, or 2 years.

In some embodiments, each of the agents is administered at about thesame dose as the dose used for monotherapy. In other embodiments, theTNF-α blocking agent is administered at a dosage that is equal to orless than an amount required for efficacy if administered alone (e.g.,at least 10, 20, 30, or 40% less). Likewise, the TWEAK blocking agentcan be administered at a dosage that is equal to or less than an amountrequired for efficacy if administered alone (e.g., at least 10, 20, 30,or 40% less). For example, in some embodiments in which the subject haspreviously received the TNF-α blocking agent, the subject isadministered a reduced dose of the TNF-α blocking agent after receivingthe TWEAK blocking agent (relative to the dose of the TNF-α blockingagent received before receiving the TWEAK blocking agent for the firsttime). The same or a different TNF-α blocking agent can be used in thecombination as was used in the previous monotherapy.

A subject can be evaluated after receiving the first and second agent,e.g., for indicia of responsiveness. A skilled artisan can use variousclinical or other indicia of effectiveness of treatment. The subject canbe monitored at various times during a regimen.

In one embodiment, the TWEAK blocking agent and the TNF-α blocking agentare administered in amounts effective to inhibit the collective effectsof TWEAK and TNF-α pathways in cells that generate inflammatory signals,e.g., synoviocytes, chondrocytes, osteoclasts, osteoblasts, dermalfibroblasts, monocytes, macrophages, or endothelial cells. The agentscan be administered in amounts effective to reduce transcription of aset of genes induced by TWEAK and TNF-α in such cells, e.g., to reducetranscription of genes synergistically activated by TWEAK and TNF-α,e.g., one or more genes list in Table 1 in synoviocytes, chondrocytes,osteoclasts, or osteoblasts.

In some embodiments, the TWEAK blocking agent is administered in anamount that is at least 20, 30, 50, 60, or 70% less than standarddosages for TWEAK blocking agent monotherapy (or a TWEAK blocking agenttherapy in the absence of TNF-α blocking agent) for treating an adultsubject for rheumatoid arthritis. For example, the TWEAK blocking agentis administered in an amount less than that required to be effective asa monotherapy.

In some embodiments, the TNF-α blocking agent is administered in anamount that is at least 20, 30, 50, 60, or 70% less than standarddosages for a TNF-α blocking agent monotherapy (or a TNF-α blockingagent therapy in the absence of a TWEAK blocking agent) for treating anadult subject for rheumatoid arthritis. For example, the TNF-α blockingagent is administered in an amount less than that required to beeffective as a monotherapy. In other embodiments, the TNF-α blockingagent and the TWEAK blocking agent are administered in the same dose asthat used in monotherapy.

For example, the subject is not receiving methotrexate. In oneembodiment, the subject is not receiving any other disease modifyinganti-rheumatic drug (DMARD), i.e., other than the TWEAK blocking agentand the TNF-α blocking agent.

The amounts can be sufficient to result in a statistically significantreduction in joint damage as measured by the Sharp erosion score. Forexample, the subject can be monitored at one or more instances for aparameter indicative of the disorder.

The method can include evaluating (e.g., monitoring one or times, e.g.,periodically) the subject, e.g., for symptoms of the disorder or indiciathat grade disorder severity. For example, in the case of rheumatoidarthritis, it is possible to use the total Sharp score (TSS), Sharperosion score, HAQ disability index, or radiological method.

In another aspect, the disclosure features a method that includes:administering, to a subject (e.g., a human subject) who has or is atrisk for rheumatoid arthritis, a TWEAK blocking agent in combinationwith another DMARD (e.g., a biologic DMARD), in amounts and for a timeto provide an overall therapeutic effect. Some examples of DMARDs fortreating rheumatoid arthritis are described herein.

In another aspect, the invention features a method of reducing jointinflammation in a subject in need thereof. The method includesadministering to a subject who suffers from joint inflammation a TNF-αblocking agent in combination with a TWEAK blocking agent, e.g., asdescribed herein. In some cases, the subject has an arthritic disorder,e.g., rheumatoid arthritis.

Also featured is a pharmaceutical composition that includes: a TWEAKblocking agent; and a DMARD, e.g., a TNF-α blocking agent or otherDMARD.

Kits can also be provided that include a TWEAK blocking agent and aDMARD (e.g., a TNF-α blocking agent or other DMARD). The agents can beprovided as separate pharmaceutical compositions or a singlepharmaceutical composition. The kit can further include instructions foradministration to treat rheumatoid arthritis, a device for administeringthe agents, and/or reagents for evaluating a parameter, e.g., a clinicalparameter associated with the disorder.

In another aspect, the disclosure features a method that includes:identifying a subject who has inflammation mediated by TWEAK and TNF-α,and/or increased TWEAK expression or activity, and/or increasedexpression or activity of a biomarker whose expression is modulated(e.g., increased) by TWEAK (see, e.g., Table 2); and administering tothe subject a therapy. For example, therapy can include administering:(i) a TWEAK blocking agent; (ii) a TNF-α blocking agent; or (iii) acombination of (i) and (ii). A “TWEAK/TNF-α synergistically activatedcellular program” is a cellular state characterized by properties thatresult from stimulation by particular doses of both TWEAK and TNF-α, butwhich are not attained to a comparable degree by stimulation with thatdose of TWEAK in the absence of that dose of TNF-α nor by stimulation bythat dose TNF-α in the absence of that dose of TWEAK. The subject can beidentified by evaluating expression of one or more genes in cells thatgenerate inflammatory signals, e.g., synoviocytes, chondrocytes,osteoclasts, osteoblasts, or dermal fibroblasts, or associated tissue,obtained from the subject. The one or more genes from Table 1 can beevaluated. The subject can also be evaluated for one or more symptoms ofrheumatoid arthritis.

In another aspect, the disclosure features a method that includes:administering, to a human subject who has or is at risk for rheumatoidarthritis, and who is being or has been withdrawn from a DMARD (otherthan a TWEAK blocking agent), a TWEAK blocking agent, e.g., in an amountand for a time effective to provide an overall therapeutic effect. Themethod can be used to treat a subject has not previously received aTWEAK blocking agent or who has not recently received a TWEAK blockingagent, e.g., within the last month, six months, or year.

In one embodiment, the DMARD that is being or has been withdrawn is aTNF-α blocking agent. The subject may have an inadequate response to theTNF-α blocking agent. As used herein, an “inadequate response” refers toa response that, as assessed by a patient or a skilled clinician,exhibits insufficient efficacy or intolerable or unacceptable toxicity.Insufficient efficacy can be defined by failure to meet a predeterminedlevel of response to treatment. For example, the TNF-α blocking agentmay cause toxicity, induce an immune-compromised state, or lacksefficacy, thereby prompting its withdrawal. For example, the subject isrefractory to therapy with the TNF-α blocking agent. The subject mayhave, e.g., tuberculosis, an opportunistic infection,glomerulonephritis, a demyelinating syndrome, a lupus-like reaction, ora pathogenic bacterial infection. In some cases, an inadequate responseis indicated by an adverse event detected during treatment with theTNF-α blocking agent.

The TNF-α blocking agent may have been administered within the previousyear, three months, month, two weeks, or week. In some cases, thesubject may still be administered the TNF-α blocking agent, but itsdosage may be reduced or may be a final dosage, e.g., a dosage providedprior to complete termination. In other cases, administration of theTNF-α blocking agent is ceased such that, upon administration of one ormore doses of the TWEAK blocking agent, the subject is no longerreceiving the TNF-α blocking agent.

In other embodiments, the DMARD that is being or has been withdrawn ismethotrexate, parenteral gold, sulphasalazine, or hydroxychloroquinone.For example, the DMARD is other than a TNF-α blocking agent. The DMARDcan be withdrawn due to toxicity, immune suppression or lack ofefficacy. For example, an adverse event may be detected during treatmentwith the DMARD.

In another aspect, the disclosure features a method that includes:detecting an adverse event in a human subject who has rheumatoidarthritis, and is being treated with a DMARD other than a TWEAK blockingagent; and administering, to the subject, a TWEAK blocking agent in anamount and for a time effective to provide an overall therapeuticeffect.

In one embodiment, the subject is being treated with a TNF-α blockingagent. The method can further include withdrawing the TNF-α blockingagent. The adverse event can include a lupus-like reaction, a bacterialor opportunistic infection, or tuberculosis.

In one aspect, the disclosure features a method of treating a subjectfor an inflammatory disorder, particularly one that a TNF-α blockingagent does not exacerbate. The inflammatory disorder can be rheumatoidarthritis, or a disorder other than rheumatoid arthritis. For example,the disorder can be psoriatic arthritis, ankylosing spondylitis,inflammatory bowel disease (including ulcerative colitis and Crohn'sdisease), psoriasis, or inflammatory myositis. Still other examples ofinflammatory disorders include Langerhans-cell histiocytosis, adultrespiratory distress syndrome/bronchiolitis obliterans, Wegener'sgranulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonaryfibrosis, dermatomyositis or polymyositis, non-infectious scleritis,chronic sarcoidosis with pulmonary involvement, myelodysplasticsyndromes/refractory anemia with excess blasts, ulcerative colitis,moderate to severe chronic obstructive pulmonary disease, and giant cellarteritis. The method includes administering, to a human subject who hasor is at risk for an inflammatory disorder, a TWEAK blocking agent in anamount and for a time to provide an overall therapeutic effect. Themethod can include administering the TWEAK blocking agent in combinationwith a TNF-α blocking agent, in amounts and for a time to provide anoverall therapeutic effect, or administering the TWEAK blocking agentwithout providing (e.g., withholding) the TNF-α blocking agent. In oneembodiment, the subject is less than 17 years of age, and the disorderis juvenile rheumatoid arthritis or pediatric psoriasis. The method caninclude other features described herein.

In another aspect, the disclosure features a method of evaluating a testcompound, e.g., for ability to modulate a TWEAK and/or TNF-α response invitro or in vivo. A TWEAK response includes modulation of TWEAK itselfor modulation of a TWEAK receptor. The method includes contacting thetest compound to a cell, tissue, or organism, in the presence of TWEAKand/or TNF-α, e.g., exogenous TWEAK and/or TNF-α. The method furtherincludes evaluating whether the test compound modulates ability of thecell, tissue, or organism to respond to TWEAK and/or TNF-α, e.g., toreduce TWEAK/TNF-α mediated cellular programs. The method can includeevaluating expression or activity of one or more genes in Table 1 orTable 2. The method can further include evaluating ability of the testcompound to modulate a disorder, e.g., using an animal model of a humandisorder described herein.

In another aspect, the disclosure features a method of evaluating asubject, e.g., a human subject. The subject can be evaluated in advanceof providing one or more agents described herein, while receiving one ormore such agents, or after receiving one or more such agents. The methodincludes evaluating cells (e.g., in a sample obtained from the subject),tissue or other material from the subject to determine if expression(including protein and mRNA expression) of one or more genes in Table 2are altered relative to a reference value. The reference value can be avalue associated with a reference value for a normal subject, a controlsubject, or a value determined, e.g., for a cohort of subjects. Thereference value can be a reference value for the subject him or herself,e.g., at another instance, e.g., before receiving one or more agents,and so forth. The information from the evaluating can be stored on acomputer-readable medium or another medium, and/or communicated, e.g.,using a computer network. The method can be used to determine if thepatient is or is predicted to be TWEAK responsive. For example, apatient that has an elevated level of expression of one or more genes inTable 2 can be indicated to be TWEAK responsive. The method can includeproviding an indication that the subject is TWEAK responsive, andoptionally instructions to administer a TWEAK blocking agent. The methodcan further include administering the TWEAK blocking agent.

In another aspect, the disclosure features a medicament comprising aTWEAK blocking agent and a TNF-α blocking agent, e.g., for use intherapy.

In another aspect, the disclosure features use of a TWEAK blocking agentand a TNF-α blocking agent for the preparation of a medicament, e.g.,for the treatment of an inflammatory disorder described herein, e.g.,joint inflammation or an arthritic disorder, e.g., rheumatoid arthritis.

In another aspect, the disclosure features use of a TWEAK blocking agentfor the preparation of a medicament, e.g., for the treatment of aninflammatory disorder described herein, e.g., joint inflammation or anarthritic disorder, e.g., rheumatoid arthritis in subjects who areunresponsive to therapy with another DMARD.

All patents, patent applications, and references cited herein are herebyincorporated by reference in their entireties. In the case of conflict,the present application controls.

The term “synergy” refers to a result from at least two events that isgreater than the sum of the result of each event individually. ANOVAscan be used to determine a synergy factor in the following equation:

R=A+B+ε(A*B)

Exemplary values for the synergy factor ε can be greater than zero or apredetermined value, e.g., 1, 2, or more.

The term “treating” refers to administering a therapy in an amount,manner, and/or mode effective to improve or prevent a condition,symptom, or parameter associated with a disorder or to prevent onset,progression, or exacerbation of the disorder, to either a statisticallysignificant degree or to a degree detectable to one skilled in the art.Accordingly, treating can achieve therapeutic and/or prophylacticbenefits. An effective amount, manner, or mode can vary depending on thesubject and may be tailored to the subject.

Reference to inhibition includes at least partial inhibition as well asother degrees of inhibition, e.g., substantial or complete.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a line graph showing average arthritis index scores in a mCIAmodel of arthritis in mice that were treated with a combination of TWEAKand TNF-α blocking agents, a TWEAK blocking agent alone, a TNF-αblocking agent alone, a PBS control, or isotype-matched controls.

FIG. 2 is a plot showing average metatarsal height values in a mCIAmodel of arthritis in mice that were treated with a combination of TWEAKand TNF-α blocking agents, a TWEAK blocking agent alone, a TNF-αblocking agent alone, a PBS control, or isotype-matched controls.

FIG. 3 is a line graph showing percent body weight change in a mCIAmodel of arthritis in mice that were treated with a combination of TWEAKand TNF-α blocking agents, a TWEAK blocking agent alone, a TNF-αblocking agent alone, a PBS control, or isotype-matched controls.

FIG. 4 depicts two line plots showing average arthritis index values inCIA models of arthritis in animals treated with anti-TWEAK blockingantibodies (anti-TWEAK mAbs) or controls. The left panel shows resultsobtained using a mouse CIA model; the right panel shows results obtainedusing a rat CIA model.

FIG. 5 depicts two line plots showing average arthritis index values inCIA models of arthritis in animals treated with anti-TWEAK blockingantibodies (anti-TWEAK mAbs) or controls. The figures show the resultsof two dosing regimens: in the first, the antibody is administered atthe time of arthritis induction, in the second, the antibody isadministered after arthritis induction. The left panel shows resultsobtained using a mouse CIA model; the right panel shows results obtainedusing a rat CIA model.

FIG. 6 includes five bar graphs showing levels of inflammation andcartilage and bone loss in a rat CIA model of arthritis in rats treatedwith anti-TWEAK blocking antibodies (ABG. 11) or controls. Similarlyfindings can be observed in a mouse model.

FIG. 7 is a plot showing serum TWEAK levels at various time points afterinduction of arthritis in a mCIA model and in a control mouse (DBA/1).

FIG. 8 includes four bar graphs showing the levels of MMP9,lymphotactin, IP-10, and IL-6 at various time points after induction ofarthritis in the mCIA model in mice treated with anti-TWEAK blockingantibodies (P5G9 and P5G9 (Full)) or controls.

DETAILED DESCRIPTION

We have discovered that the TWEAK and TNF-α pathways independentlycontribute to inflammatory responses, e.g., in synovial cells present injoints, and that both TWEAK and TNF-α can independently activate similarsets of genes indicating redundancy between the two pathways.Accordingly, reducing the activity of both pathways provides anadvantageous therapeutic route to ameliorating inflammation, e.g., injoints, e.g., in arthritic conditions. Concurrent blocking of both TWEAKand TNF-α pathways proved beneficial in a mouse model of rheumatoidarthritis (mCIA) and achieved results greater than blocking one of thesepathways.

In addition to rheumatoid arthritis, reducing activity of both pathwaysmay be used to treat in other disorders, e.g., other inflammatorydisorders such as psoriatic arthritis, ankylosing spondylitis,inflammatory bowel disease, psoriasis, inflammatory myositis, and otherdisorders disclosed herein. A variety of methods can be used to reduceactivity of the TWEAK and TNF-α pathways. For example, it is possible toadminister a TWEAK blocking agent in combination with a TNF-α blockingagent. Examples of these and other agents are further described below.

In some implementations, therapeutic benefit can be achieved by reducingone of the two pathways. For example, a TWEAK blocking agent can beadministered to a subject who has an inadequate response to atherapeutic that modulates just one of the pathways, e.g., an inadequateresponse to a TNF-α blocking agent or an inadequate response to a TWEAKblocking agent. A TWEAK blocking agent can also be administered to asubject who is or who is planning to withdraw from a DMARD treatmentwith another agent, e.g., an agent other than a TNF-α blocking agent.

TWEAK Blocking Agents

A variety of agents can be used as a TWEAK blocking agent. The agent maybe any type of compound (e.g., small organic or inorganic molecule,nucleic acid, protein, or peptide mimetic) that can be administered to asubject. In one embodiment, the blocking agent is a biologic, e.g., aprotein having a molecular weight of between 5-300 kDa. For example, aTWEAK blocking agent may inhibit binding of TWEAK to a TWEAK receptor ormay prevent TWEAK-mediated NF-κB activation. A typical TWEAK blockingagent can bind to TWEAK or a TWEAK receptor, e.g., Fn14. A TWEAKblocking agent that binds to TWEAK or a TWEAK receptor may alter theconformation of TWEAK or a TWEAK receptor, block the binding site onTWEAK or a TWEAK receptor, or otherwise decrease the affinity of TWEAKfor a TWEAK receptor or prevent the interaction between TWEAK and aTWEAK receptor.

A TWEAK blocking agent (e.g., an antibody) may bind to TWEAK or to aTWEAK receptor with a K_(d) of less than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, or10⁻¹⁰ M. In one embodiment, the blocking agent binds to TWEAK with anaffinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better thanits affinity for TNF-α or another TNF superfamily member (other thanTWEAK). In one embodiment, the blocking agent binds to the TWEAKreceptor with an affinity at least 5, 10, 20, 50, 100, 200, 500, or1000-fold better than its affinity for the TNF receptor or a receptorfor another TNF superfamily member. A preferred TWEAK blocking agentspecifically binds TWEAK or TWEAK-R.

Exemplary TWEAK protein molecules include human TWEAK (e.g., AAC51923,shown as SEQ ID NO:1)), mouse TWEAK (e.g., NP_(—)035744.1), rat TWEAK(e.g., XP_(—)340827.1), and Pan troglodytes TWEAK (e.g.,XP_(—)511964.1). Also included are proteins that include an amino acidsequence at least 90, 92, 95, 97, 98, 99% identical or completelyidentical to the mature processed region of the aforementioned TWEAKproteins (e.g., an amino acid sequence at least 90, 92, 95, 97, 98, 99%identical or completely identical to amino acids X₁-249 of SEQ ID NO:1,where amino acid X₁ is selected from the group of residues 75-115 of SEQID NO:1, e.g., X₁ is residue Arg 93 of SEQ ID NO:1) and proteins encodedby a nucleic acid that hybridizes under high stringency conditions to ahuman, mouse, rat, or Pan troglodytes gene encoding a naturallyoccurring TWEAK protein. Preferably, a TWEAK protein, in its processedmature form, is capable of providing at least one TWEAK activity, e.g.,ability to activate Fn14.

Exemplary Fn14 protein molecules include human Fn14 (e.g.,NP_(—)057723.1, shown as SEQ ID NO:2), mouse Fn14 (e.g.,NP_(—)038777.1), and rat Fn14 (e.g., NP_(—)851600.1) as well as solubleproteins that include an amino acid sequence at least 90, 92, 95, 97,98, 99% identical or completely identical to the extracellular domain ofFn14 (and TWEAK-binding fragments thereof) and proteins encoded by anucleic acid that hybridizes under high stringency conditions to ahuman, mouse, rat, or Pan troglodytes gene encoding a naturallyoccurring Fn14 protein. Preferably, a Fn14 protein useful in the methodsdescribed herein is a soluble Fn14 (lacking a transmembrane domain) thatincludes a region that binds to a TWEAK protein, e.g., an amino acidsequence at least 90, 92, 95, 97, 98, or 99% identical, or completelyidentical, to amino acids 28-X₁ of SEQ ID NO:2, where amino acid X₁ isselected from the group of residues 68 to 80 of SEQ ID NO:2.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences. Alignments of relatedproteins described herein are instructive for identifying amino acidpositions that tolerate modification, e.g., insertion, deletion, andsubstitution, e.g., conservative or non-conservative substitution.

As used herein, the term “hybridizes under high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. High stringency hybridizationconditions include hybridization in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 65° C., or substantiallysimilar conditions.

Exemplary TWEAK blocking agents include antibodies that bind to TWEAK orTWEAK-R and soluble forms of the TWEAK-R that compete with cell surfaceTWEAK-R for binding to TWEAK. An example of a soluble form of theTWEAK-R is an Fc fusion protein that includes at least a portion of theextracellular domain of TWEAK-R (e.g., a soluble TWEAK-binding fragmentof TWEAK-R), referred to as TWEAK-R-Fc. Other soluble forms of TWEAK-R,e.g., forms that do not include an Fc domain, can also be used. Antibodyblocking agents are further discussed below.

Other types of blocking agents, e.g., small molecules, nucleic acid ornucleic acid-based aptamers, and peptides, can be isolated by screening,e.g., as described in Jhaveri et al. Nat. Biotechnol. 18:1293 and U.S.Pat. No. 5,223,409. Exemplary assays for determining if an agent bindsto TWEAK or TWEAK-R and for determining if an agent modulates aTWEAK/TWEAK-R interaction are described, e.g., in U.S. 2004-0033225.

An exemplary soluble form of the TWEAK-R protein includes a region ofthe TWEAK-R protein that binds to TWEAK, e.g., about amino acids 32-75,31-75, 31-78, or 28-79 of SEQ ID NO:2. This region can be physicallyassociated, e.g., fused to another amino acid sequence, e.g., an Fcdomain, at its N- or C-terminus. The region from TWEAK-R can be spacedby a linker from the heterologous amino acid sequence. U.S. Pat. No.6,824,773 describes an exemplary TWEAK-R fusion protein.

TNF-α Blocking Agents

A variety of agents can be used as a TNF-α blocking agent. The agent maybe any type of compound (e.g., small organic or inorganic molecule,nucleic acid, protein, or peptide mimetic) that can be administered to asubject. In one embodiment, the blocking agent is a biologic, e.g., aprotein having a molecular weight of between 5-300 kDa. For example, aTNF-α blocking agent may inhibit binding of TNF-α to a TNF-α receptor orotherwise prevent TNF-α receptor downstream signalling. A typical TNF-αblocking agent can bind to TNF-α or a TNF-α receptor, e.g., TNFR-I orTNFR-II. A TNF-α blocking agent that binds to TNF-α or a TNF-α receptormay alter the conformation of TNF-α or a TNF-α receptor, block thebinding site on TNF-α or a TNF-α receptor, or otherwise decrease theaffinity of TNF-α for a TNF-α receptor or prevent the interactionbetween TNF-α and a TNF-α receptor.

A TNF-α blocking agent (e.g., an antibody) may bind to TNF-α or to aTNF-α receptor with a K_(d) of less than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, or10⁻¹⁰ M. In one embodiment, the blocking agent binds to TNF-α with anaffinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better thanits affinity for TWEAK or another TNF superfamily member (other thanTNF-α). A preferred TNF-α blocking agent specifically binds TNF-α or aTNF-α-R, such as a TNF-α or TNF-α-R specific antibody.

Exemplary TNF-α blocking agents include antibodies that bind to TNF-α orTNF-α-R and soluble forms of the TNF-α-R that compete with cell surfaceTNF-α-R for binding to TNF-α. An example of a soluble form of theTNF-α-R is an Fc fusion protein that includes at least a portion of theextracellular domain of TNF-α-R (e.g., a soluble TNF-α-binding fragmentof TNF-α-R), referred to as TNF-α-R-Fc. Other soluble forms of TNF-α-R,e.g., forms that do not include an Fc domain, can also be used. Antibodyblocking agents are further discussed below.

An exemplary soluble form of a TNF-α receptor protein is ENBREL®. Seee.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med.(1996) Vol. 44, 235A. U.S. Pat. No. 6,572,852 describes additionalexamples. The recommended dose of ENBREL® for adult patients withrheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis is50 mg per week given as one subcutaneous (SC) injection using a 50 mg/mLsingle-use prefilled syringe. In addition to ENBREL®, other similarand/or corresponding regions of TNF-α receptors can be physicallyassociated, e.g., fused to another amino acid sequence, e.g., an Fcdomain, at its N- or C-terminus.

Other well characterized examples of TNF-α blocking agents include:infliximab (REMICADE®), a chimeric antibody that binds to tumor necrosisfactor-alpha (TNF-α) and adalimumab (HUMIRA®), a human antibody thatbinds to TNF-α. For example, the recommended dose of REMICADE® is 3mg/kg given as an intravenous infusion followed with additional similardoses at 2 and 6 weeks after the first infusion then every 8 weeksthereafter.

Additional examples of TNF-α blocking agents include chimeric,humanized, human or in vitro generated antibodies (or antigen-bindingfragments thereof) to TNF (e.g., human TNF-α), such as D2E7, (humanTNF-α antibody, U.S. Pat. No. 6,258,562; BASF),CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNF-α antibody;Celltech/Pharmacia), cA2 (chimeric anti-TNFα antibody; REMICADE™,Centocor, also mentioned above); anti-TNF antibody fragments (e.g.,CPD870); soluble fragments of the TNF receptors, e.g., p55 or p75 humanTNF receptors or derivatives thereof, e.g., 75 kd TNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL™), p55 kd TNFR-IgG (55 kD TNFreceptor-IgG fusion protein (LENERCEPT™)); enzyme antagonists, e.g.,TNFα converting enzyme (TACE) inhibitors (e.g., an alpha-sulfonylhydroxamic acid derivative, PCT Application WO 01/55112, andN-hydroxyformamide TACE inhibitor GW 3333, -005, or -022); andTNF-bp/s-TNFR (soluble TNF binding protein; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer. J.Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42).

Antibodies

Exemplary TWEAK blocking agents include antibodies that bind to TWEAKand/or TWEAK-R. In one embodiment, the antibody inhibits the interactionbetween TWEAK and a TWEAK receptor, e.g., by physically blocking theinteraction, decreasing the affinity of TWEAK and/or TWEAK-R for itscounterpart, disrupting or destabilizing TWEAK complexes, sequesteringTWEAK or a TWEAK-R, or targeting TWEAK or TWEAK-R for degradation. Inone embodiment, the antibody can bind to TWEAK or TWEAK-R at one or moreamino acid residues that participate in the binding interface betweenTWEAK and its receptor. Such amino acid residues can be identified,e.g., by alanine scanning. In another embodiment, the antibody can bindto residues that do not participate in the binding interface. Forexample, the antibody can alter a conformation of TWEAK or TWEAK-R andthereby reduce binding affinity, or the antibody may sterically hinderbinding. In one embodiment, the antibody can prevent activation of aTWEAK-R mediated event or activity (e.g., NF-κB activation).

Similarly, exemplary TNF-α blocking agents include antibodies that bindto TNF-α and/or a TNF-α receptor, e.g., TNFR-I or TNFR-II. In oneembodiment, the antibody inhibits the interaction between TNF-α and aTNF-α receptor, e.g., by physically blocking the interaction, decreasingthe affinity of TNF-α and/or TNF-α-R for its counterpart, disrupting ordestabilizing TNF-α complexes, sequestering TNF-α or a TNF-α receptor,or targeting TNF-α or TNF-α receptor for degradation. In one embodiment,the antibody can bind to TNF-α or TNF-α receptor at one or more aminoacid residues that participate in the TNF-α/TNF-α receptor bindinginterface. Such amino acid residues can be identified, e.g., by alaninescanning. In another embodiment, the antibody can bind to residues thatdo not participate in the TNF-α/TNF-α receptor binding. For example, theantibody can alter a conformation of TNF-α or TNF-α receptor and therebyreduce binding affinity, or the antibody may sterically hinderTNF-α/TNF-α receptor binding.

As used herein, the term “antibody” refers to a protein that includes atleast one immunoglobulin variable region, e.g., an amino acid sequencethat provides an immunoglobulin variable domain or an immunoglobulinvariable domain sequence. For example, an antibody can include a heavy(H) chain variable region (abbreviated herein as VH), and a light (L)chain variable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab fragments,F(ab′)₂ fragments, Fd fragments, Fv fragments, and dAb fragments) aswell as complete antibodies, e.g., intact and/or full lengthimmunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE,IgD, IgM (as well as subtypes thereof). The light chains of theimmunoglobulin may be of types kappa or lambda. In one embodiment, theantibody is glycosylated. An antibody can be functional forantibody-dependent cytotoxicity and/or complement-mediated cytotoxicity,or may be non-functional for one or both of these activities.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the FR's and CDR's has been preciselydefined (see, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; and Chothia, C. et al.(1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein.Each VH and VL is typically composed of three CDR's and four FR's,arranged from amino-terminus to carboxyl-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

An “immunoglobulin domain” refers to a domain from the variable orconstant domain of immunoglobulin molecules. Immunoglobulin domainstypically contain two β-sheets formed of about seven β-strands, and aconserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay(1988) Ann. Rev Immunol. 6:381-405). An “immunoglobulin variable domainsequence” refers to an amino acid sequence that can form a structuresufficient to position CDR sequences in a conformation suitable forantigen binding. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may omit one, two, or more N- or C-terminal aminoacids, internal amino acids, may include one or more insertions oradditional terminal amino acids, or may include other alterations. Inone embodiment, a polypeptide that includes an immunoglobulin variabledomain sequence can associate with another immunoglobulin variabledomain sequence to form a target binding structure (or “antigen bindingsite”), e.g., a structure that interacts with a target protein, e.g.,TWEAK, a TWEAK receptor, TNF-α, TNFR-I, or TNFR-II.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains. The heavy and light immunoglobulin chains can be connected bydisulfide bonds. The heavy chain constant region typically includesthree constant domains, CH1, CH2, and CH3. The light chain constantregion typically includes a CL domain. The variable region of the heavyand light chains contains a binding domain that interacts with anantigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

One or more regions of an antibody can be human, effectively human, orhumanized. For example, one or more of the variable regions can be humanor effectively human. For example, one or more of the CDRs, e.g., HCCDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, can be human.Each of the light chain CDRs can be human. HC CDR3 can be human. One ormore of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4of the HC or LC. In one embodiment, all the framework regions are human,e.g., derived from a human somatic cell, e.g., a hematopoietic cell thatproduces immunoglobulins or a non-hematopoietic cell. In one embodiment,the human sequences are germline sequences, e.g., encoded by a germlinenucleic acid. One or more of the constant regions can be human,effectively human, or humanized. In another embodiment, at least 70, 75,80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1, FR2, andFR3, collectively, or FR1, FR2, FR3, and FR4, collectively) or theentire antibody can be human, effectively human, or humanized. Forexample, FR1, FR2, and FR3 collectively can be at least 70, 75, 80, 85,90, 92, 95, 98, or 99% identical, or completely identical, to a humansequence encoded by a human germline segment.

An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

A “humanized” immunoglobulin variable region is an immunoglobulinvariable region that is modified such that the modified form elicitsless of an immune response in a human than does the non-modified form,e.g., is modified to include a sufficient number of human frameworkamino acid positions such that the immunoglobulin variable region doesnot elicit an immunogenic response in a normal human. Descriptions of“humanized” immunoglobulins include, for example, U.S. Pat. Nos.6,407,213 and 5,693,762. In some cases, humanized immunoglobulins caninclude a non-human amino acid at one or more framework amino acidpositions.

Antibody Generation

Antibodies that bind to a target protein (e.g., TWEAK, TWEAK-R, TNF-α,TNFR-I or TNFR-II) can be generated by a variety of means, includingimmunization, e.g., using an animal, or in vitro methods such as phagedisplay. All or part of the target protein can be used as an immunogenor as a target for selection. In one embodiment, the immunized animalcontains immunoglobulin producing cells with natural, human, orpartially human immunoglobulin loci. In one embodiment, the non-humananimal includes at least a part of a human immunoglobulin gene. Forexample, it is possible to engineer mouse strains deficient in mouseantibody production with large fragments of the human Ig loci. Using thehybridoma technology, antigen-specific monoclonal antibodies derivedfrom the genes with the desired specificity may be produced andselected. See, e.g., XENOMOUSE™, Green et al. (1994) Nat. Gen. 7:13-21;U.S. 2003-0070185; U.S. Pat. No. 5,789,650; and PCT Application WO96/34096.

Non-human antibodies to the target proteins can also be produced, e.g.,in a rodent. The non-human antibody can be humanized, e.g., as describedin EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761; and 6,407,213,deimmunized, or otherwise modified to make it effectively human.

EP 239 400 (Winter et al.) describes altering antibodies by substitution(within a given variable region) of their complementarity determiningregions (CDRs) for one species with those from another. Typically, CDRsof a non-human (e.g., murine) antibody are substituted into thecorresponding regions in a human antibody by using recombinant nucleicacid technology to produce sequences encoding the desired substitutedantibody. Human constant region gene segments of the desired isotype(usually gamma I for CH and kappa for CL) can be added and the humanizedheavy and light chain genes can be co-expressed in mammalian cells toproduce soluble humanized antibody.

Other methods for humanizing antibodies can also be used. For example,other methods can account for the three dimensional structure of theantibody, framework positions that are in three dimensional proximity tobinding determinants, and immunogenic peptide sequences. See, e.g., PCTApplication WO 90/07861; U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089;and 5,530,101; Tempest et al. (1991) Biotechnology 9:266-271 and U.S.Pat. No. 6,407,213. Still another method is termed “humaneering” and isdescribed, for example, in U.S. 2005-008625.

Fully human monoclonal antibodies that bind to target proteins can beproduced, e.g., using in vitro-primed human splenocytes, as described byBoerner et al. (1991) J. Immunol. 147:86-95. They may be prepared byrepertoire cloning as described by Persson et al. (1991) Proc. Nat.Acad. Sci. USA 88:2432-2436 or by Huang and Stollar (1991) J. Immunol.Methods 141:227-236; also U.S. Pat. No. 5,798,230. Large non-immunizedhuman phage display libraries may also be used to isolate high affinityantibodies that can be developed as human therapeutics using standardphage technology (see, e.g., Hoogenboom et al. (1998) Immunotechnology4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-378; and U.S.2003-0232333).

Antibody and Protein Production

Antibodies and other proteins described herein can be produced inprokaryotic and eukaryotic cells. In one embodiment, the antibodies(e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g.,Powers et al. (2001) J. Immunol. Methods 251:123-35), Hanseula, orSaccharomyces.

Antibodies, particularly full length antibodies, e.g., IgG's, can beproduced in mammalian cells. Exemplary mammalian host cells forrecombinant expression include Chinese Hamster Ovary (CHO cells)(including dhfr− CHO cells, described in Urlaub and Chasin (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621),lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COScells, K562, and a cell from a transgenic animal, e.g., a transgenicmammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the immunoglobulindomain, the recombinant expression vectors may carry additional nucleicacid sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216; 4,634,665; and 5,179,017). Exemplary selectable markergenes include the dihydrofolate reductase (DHFR) gene (for use in dhfr⁻host cells with methotrexate selection/amplification) and the neo gene(for G418 selection).

In an exemplary system for recombinant expression of an antibody (e.g.,a full length antibody or an antigen-binding portion thereof), arecombinant expression vector encoding both the antibody heavy chain andthe antibody light chain is introduced into dhfr− CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, to transfect the host cells, to selectfor transformants, to culture the host cells, and to recover theantibody from the culture medium. For example, some antibodies can beisolated by affinity chromatography with a Protein A or Protein G.

Antibodies (and Fc fusions) may also include modifications, e.g.,modifications that alter Fc function, e.g., to decrease or removeinteraction with an Fc receptor or with Clq, or both. For example, thehuman IgG1 constant region can be mutated at one or more residues, e.g.,one or more of residues 234 and 237, e.g., according to the numbering inU.S. Pat. No. 5,648,260. Other exemplary modifications include thosedescribed in U.S. Pat. No. 5,648,260.

For some proteins that include an Fc domain, the antibody/proteinproduction system may be designed to synthesize antibodies or otherproteins in which the Fc region is glycosylated. For example, the Fcdomain of IgG molecules is glycosylated at asparagine 297 in the CH2domain. The Fc domain can also include other eukaryoticpost-translational modifications. In other cases, the protein isproduced in a form that is not glycosylated.

Antibodies and other proteins can also be produced by a transgenicanimal. For example, U.S. Pat. No. 5,849,992 describes a method forexpressing an antibody in the mammary gland of a transgenic mammal. Atransgene is constructed that includes a milk-specific promoter andnucleic acid sequences encoding the antibody of interest, e.g., anantibody described herein, and a signal sequence for secretion. The milkproduced by females of such transgenic mammals includes,secreted-therein, the protein of interest, e.g., an antibody or Fcfusion protein. The protein can be purified from the milk, or for someapplications, used directly.

Methods described in the context of antibodies can be adapted to otherproteins, e.g., Fc fusions and soluble receptor fragments.

Nucleic Acid Blocking Agents

In certain implementations, nucleic acid blocking agents are used todecrease expression of a target protein such as TWEAK, a TWEAK-R (e.g.,Fn14), TNF-α, TNFR-I or TNFR-II. These agents can be used in place of orin addition to proteinaceous TWEAK blocking agents and TNF-α blockingagents. In one embodiment, the nucleic acid antagonist is an siRNA thatis directed against the mRNA produced from an endogenous gene thatencodes the target protein. For example, the siRNA includes a regioncomplementary to the mRNA. Other types of antagonistic nucleic acids canalso be used, e.g., a dsRNA, a ribozyme, a triple-helix former, or anantisense nucleic acid.

siRNAs are small double stranded RNAs (dsRNAs) that optionally includeoverhangs. For example, the duplex region of an siRNA is about 18 to 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotidesin length. Typically, the siRNA sequences are exactly complementary tothe target mRNA. dsRNAs and siRNAs in particular can be used to silencegene expression in mammalian cells (e.g., human cells). See, e.g.,Clemens et al. (2000) Proc. Natl. Acad. Sci. USA 97:6499-6503; Billy etal. (2001) Proc. Natl. Sci. USA 98:14428-14433; Elbashir et al. (2001)Nature. 411:494-8; Yang et al. (2002) Proc. Natl. Acad. Sci. USA99:9942-9947, U.S. Pub. Apps. 2003-0166282; 2003-0143204; 2004-0038278;and 2003-0224432.

Anti-sense agents can include, for example, from about 8 to about 80nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA (e.g., an mRNAencoding a target protein) can interfere with one or more of the normalfunctions of mRNA. The functions of mRNA to be interfered with includeall key functions such as, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in by the RNA. Binding of specificprotein(s) to the RNA may also be interfered with by antisenseoligonucleotide hybridization to the RNA.

Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding a target protein. The complementary region can extend forbetween about 8 to about 80 nucleobases. The compounds can include oneor more modified nucleobases. Modified nucleobases may include, e.g.,5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, andC5-propynyl pyrimidines such as C5-propynylcytosine andC5-propynyluracil, to mention but a few. Descriptions of a variety ofnucleic acid agents are available. See, e.g., U.S. Pat. Nos. 4,987,071;5,116,742; and 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA;Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988)Nature 334:585-59; Helene, C. (1991) Anticancer Drug Des. 6:569-84;Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14:807-15.

The nucleic acids described herein, e.g., an anti-sense nucleic aciddescribed herein, can be incorporated into a gene construct to be usedas a part of a gene therapy protocol to deliver nucleic acids that canbe used to express and produce agents, e.g., anti-sense nucleic acidswithin cells. Expression constructs of such components may beadministered in any biologically effective carrier, e.g. any formulationor composition capable of effectively delivering the component gene tocells in vivo. Approaches include insertion of the subject gene in viralvectors including recombinant retroviruses, adenovirus, adeno-associatedvirus, lentivirus, and herpes simplex virus-1, or recombinant bacterialor eukaryotic plasmids. Viral vectors transfect cells directly; plasmidDNA can be delivered with the help of, for example, cationic liposomes(lipofectin) or derivatized (e.g. antibody conjugated), polylysineconjugates, gramacidin S, artificial viral envelopes or other suchintracellular carriers, as well as direct injection of the geneconstruct or CaPO₄ precipitation carried out in vivo.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo, particularly into humans. These vectors provide efficient deliveryof genes into cells, and the transferred nucleic acids are stablyintegrated into the chromosomal DNA of the host. Protocols for producingrecombinant retroviruses and for infecting cells in vitro or in vivowith such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare known to those skilled in the art. Examples of suitable packagingvirus lines for preparing both ecotropic and amphotropic retroviralsystems include *Crip, *Cre, *2 and *Am. Retroviruses have been used tointroduce a variety of genes into many different cell types, includingepithelial cells, in vitro and/or in vivo (see for example Eglitis, etal. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT ApplicationsWO 89/07136; WO 89/02468; WO 89/05345; and WO 92/07573).

Another viral gene delivery system utilizes adenovirus-derived vectors.See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeldet al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are known to those skilled in the art.

Yet another viral vector system useful for delivery of the subject geneis the adeno-associated virus (AAV). See, for example, Flotte et al.(1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989)J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol.62:1963-1973).

Artificial Transcription Factors

Artificial transcription factors can also be used to regulate expressionof a target protein, e.g., TWEAK, a TWEAK-R (e.g., Fn14), TNF-α, TNFR-Ior TNFR-II. The artificial transcription factor can be designed orselected from a library, e.g., for ability to bind to a sequence in anendogenous gene encoding target protein, e.g., in a regulatory region,e.g., the promoter. For example, the artificial transcription factor canbe prepared by selection in vitro (e.g., using phage display, U.S. Pat.No. 6,534,261) or in vivo, or by design based on a recognition code(see, e.g., PCT Application WO 00/42219 and U.S. Pat. No. 6,511,808).See, e.g., Rebar et al. (1996) Methods Enzymol 267:129; Greisman andPabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol19:656; and Wu et al. (1995) Proc. Natl. Acad. Sci. USA 92:344 for,among other things, methods for creating libraries of varied zinc fingerdomains.

Optionally, an artificial transcription factor can be fused to atranscriptional regulatory domain, e.g., an activation domain toactivate transcription or a repression domain to repress transcription.In particular, repression domains can be used to decrease expression ofendogenous genes encoding TWEAK or TWEAK-R. The artificial transcriptionfactor can itself be encoded by a heterologous nucleic acid that isdelivered to a cell or the protein itself can be delivered to a cell(see, e.g., U.S. Pat. No. 6,534,261). The heterologous nucleic acid thatincludes a sequence encoding the artificial transcription factor can beoperably linked to an inducible promoter, e.g., to enable fine controlof the level of the artificial transcription factor in the cell.

Rheumatoid Arthritis (RA)

Rheumatoid arthritis (“RA”) is a chronic inflammatory disease thatcauses pain, swelling, stiffness, and loss of function, primarily injoints. RA frequently begins in the synovium, the membrane thatsurrounds a joint creating a protective sac. In many individualssuffering from RA, leukocytes infiltrate from the circulation into thesynovium causing continuous abnormal inflammation (e.g., synovitis).Consequently, the synovium becomes inflamed, causing warmth, redness,swelling, and pain. The collagen in the cartilage is graduallydestroyed, narrowing the joint space and eventually damaging bone. Theinflammation causes erosive bone damage in the affected area. Duringthis process, the cells of the synovium grow and divide abnormally,making the normally thin synovium thick and resulting in a joint that isswollen and puffy to the touch.

As RA progresses, abnormal synovial cells can invade and destroy thecartilage and bone within the joint. The surrounding muscles, ligaments,and tendons that support and stabilize the joint can become weak andunable to work normally. RA also may cause more generalized bone lossthat may lead to osteoporosis, making bones fragile and more prone tofracture. All of these effects cause the pain, impairment anddeformities associated with RA. Regions that can be effected include thewrists, knuckles, knees and the ball of the foot. Often, many joints maybe involved, and even the spine can be affected. In about 25% of peoplewith RA, inflammation of small blood vessels can cause rheumatoidnodules, or lumps, under the skin. These are bumps under the skin thatoften form close to the joints. As the disease progresses, fluid mayalso accumulate, particularly in the ankles. Many patients with RA alsodevelop anemia, or a decrease in the normal number of red blood cells.

RA encompasses a number of disease subtypes, such as Felty's syndrome,seronegative RA, “classical” RA, progressive and/or relapsing RA, and RAwith vasculitis. Some experts classify the disease into type 1 or type2. Type 1, the less common form, lasts a few months at most and leavesno permanent disability. Type 2 is chronic and lasts for years,sometimes for life. RA can also manifest as subcutaneous rheumatoidnodules, visceral nodules, vasculitis causing leg ulcers or mononeuritismultiplex, pleural or pericardial effusions, lymphadenopathy, Felty'ssyndrome, Sjogren's syndrome, and episcleritis. These disease subtypesand also subjects showing one or more of the above symptoms can betreated using the methods described herein.

RA occurs across all races and ethnic groups. At least one geneticpredisposition has been identified and, in white populations, localizedto a pentapeptide in the HLA-DR 1 locus of class II histocompatibilitygenes.

RA can be assessed by a variety of clinical measures. Some exemplaryindicia include the total Sharp score (TSS), Sharp erosion score, andthe HAQ disability index. The methods herein can be used to achieve animprovement for at least one of these indicia.

Non-Responders to TNF-α Blocking Agents

In one aspect, subjects who have rheumatoid arthritis, or who are atrisk for RA, or who have or at risk for another disorder describedherein, can be evaluated for a parameter predictive of their ability torespond to a particular agent (e.g., a biologic DMARD), e.g., theirability to respond to a TNF-α blocking agent such as etanercept,infliximab, or adalimumab. For example, the parameter can be thepresence or absence of a nucleotide in a gene encoding TNF-α. Subjectswho are indicated to be less or non-responsive to a particular agent canbe administered an alternative agent. For example, subjects who areindicated as non-responsive to etanercept can be administered a TWEAKblocking agent.

Rheumatoid arthritis patients with the T allele of TNFα-857C/T SNP mayrespond better to etanercept therapy than homozygotes for the C allele.Kang et al. Rheumatology 2005 April; 44(4):547-52. Accordingly, RApatients that are homozygous for the C allele can be treated with aTWEAK blocking agent, and etanercept or other TNF-α blocking agent canbe withheld, or dosages can be reduced, e.g., relative to a standarddose.

Non-Responders to RA Therapies

A variety of treatments for RA, in addition to TNF-α blocking agents,are available. Many of these are therapeutics classified as diseasemodifying anti-rheumatic drugs (DMARDs). Traditional DMARDS includePLAQUENIL® (hydroxychloroquine), AZULFIDINE® (sulfasalazine) orRHEUMATREX® (methotrexate). For rheumatoid arthritis, it has beenobserved that the withdrawal rate from DMARD treatment in rheumatoidarthritis increases with the length of time the patient has beenreceiving the drug and that a number of these withdrawals relate to lossof efficacy (see, e.g., Annals of the Rheumatic Diseases (2003)62:95-96). Accordingly, a TWEAK blocking agent can also be administeredto a subject who has an inadequate response to a DMARD treatment, e.g.,an inadequate response to treatment with one of the following agents:

a. Nonsteroidal anti-inflammatory drugs including salicylates, such asaspirin.

b. Gold compounds. In some patients, gold may produce clinical remissionand decrease the formation of new bony erosions. Parenteral preparationsinclude gold sodium thiomalate or gold thioglucose. Gold should bediscontinued when signs of toxicity appear. Minor toxic manifestations(e.g., mild pruritus, minor rash) may be eliminated by temporarilywithholding gold therapy, then resuming it cautiously about 2 weeksafter symptoms have subsided. However, if toxic symptoms progress, goldshould be withheld. A TWEAK blocking agent can be administered when goldis being discontinued or when a gold chelating drug (such asdimercaprol) is being administered to counteract gold toxicity.

c. Hydroxychloroquine can also control symptoms of mild or moderatelyactive RA. Toxic effects usually are mild and include dermatitis,myopathy, and generally reversible corneal opacity. However,irreversible retinal degeneration has been reported. Hydroxychloroquinecan be withdrawn and replaced, e.g., with a TWEAK blocking agent, e.g.,upon detection of one or more of these side effects.

d. Oral penicillamine may have a benefit similar to gold. Side effectsrequiring discontinuation are more common than with gold and includemarrow suppression, proteinuria, nephrosis, other serious toxic effects(e.g., myasthenia gravis, pemphigus, Goodpasture's syndrome,polymyositis, a lupus-like syndrome), rash, and a foul taste. Oralpenicillamine can be withdrawn and replaced, e.g., with a TWEAK blockingagent, e.g., upon detection of one or more of these side effects.

e. Steroids are highly effective short-term anti-inflammatory drugs.However, their clinical benefit for RA often diminishes with time.Steroids do not predictably prevent the progression of jointdestruction. Furthermore, severe rebound often follows the withdrawal ofcorticosteroids in active disease. Accordingly, a TWEAK blocking agentcan be administered, prior to withdrawal, during withdrawal, orsubsequent to complete withdrawal. Other side effect which can triggerwithdrawal and use of a TWEAK blocking agent include peptic ulcer,hypertension, untreated infections, diabetes mellitus, and glaucoma.

f. Immunosuppressive drugs can be used in management of severe, activeRA. However, major side effects can occur, including liver disease,pneumonitis, bone marrow suppression, and, after long-term use ofazathioprine, malignancy. Withdrawal from immunosuppressive drugs caninclude administering a TWEAK blocking agent, e.g., upon detection of aside effect.

Alternatively, a TWEAK blocking agent can be administered to a subjectwho is receiving another treatment for RA, e.g., one of the abovetreatments. The combination of the treatment and the TWEAK blockingagent can be used to achieve additional therapeutic benefit and,optionally, to reduce the dosage of the other treatment. As result, sideeffects and other issues can be mitigated.

The methods described herein, e.g., a TWEAK blocking agent monotherapyor a combination therapy (such as with TWEAK and TNF-α blocking agents),can be used to treat a subject who has one or more severe complicationsof RA. Such complications include joint destruction, gastrointestinalbleeding, heart failure, pericarditis, pleuritis, lung disease, anemia,low or high platelets, eye disease, cervical (neck) spine instability,neuropathy, and vasculitis.

Other Disorders

The methods described herein can also be used to treat otherinflammatory, immune, or autoimmune disorders in patients, for exampledisorders that are not exacerbated by administration of a TNF-α blockingagent. Examples of disorders that can be treated include psoriaticarthritis, ankylosing spondylitis, inflammatory bowel disease (includingulcerative colitis and Crohn's disease), psoriasis, or inflammatorymyositis. Still other examples of inflammatory disorders includeLangerhans-cell histiocytosis, adult respiratory distresssyndrome/bronchiolitis obliterans, Wegener's granulomatosis, vasculitis,cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis orpolymyositis, non-infectious scleritis, chronic sarcoidosis withpulmonary involvement, myelodysplastic syndromes/refractory anemia withexcess blasts, ulcerative colitis, moderate to severe chronicobstructive pulmonary disease, and giant cell arteritis.

A subject who is at risk for, diagnosed with, or who has one of thesedisorders can be administered a TWEAK blocking agent in an amount andfor a time to provide an overall therapeutic effect. The TWEAK blockingagent can be administered in combination with a TNF-α blocking agent orwithout providing (e.g., withholding) the TNF-α blocking agent. In thecase of a combination therapy, the amounts and times of administrationcan be those that provide, e.g., an enhanced or synergistic therapeuticeffect. Further, the administration of the TWEAK blocking agent (with orwithout the TNF-α blocking agent) can be used as a primary, e.g., firstline treatment, or as a secondary treatment, e.g., for subjects who havean inadequate response to a previously administered therapy (i.e., atherapy other than one with a TWEAK block agent).

Pharmaceutical Compositions

A TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein,e.g., TWEAK-R-Fc) can be formulated as a pharmaceutical composition,e.g., for administration to a subject to treat a disorder describedherein, e.g., an inflammatory disorder such as rheumatoid arthritis orother disorder described herein. A TNF-α blocking agent can be similarlyformulated, either in the same composition or as a separate composition.

Typically, a pharmaceutical composition includes a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecomposition can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see e.g., Berge, S. M., etal. (1977) J. Pharm. Sci. 66:1-19).

Pharmaceutical formulation is a well-established art, and is furtherdescribed, e.g., in Gennaro (ed.), Remington: The Science and Practiceof Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN:0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) Ed., Lippincott Williams & Wilkins Publishers (1999)(ISBN: 0683305727); and Kibbe (ed.), Handbook of PharmaceuticalExcipients American Pharmaceutical Association, 3^(rd) ed. (2000) (ISBN:091733096X).

In one embodiment, the TWEAK blocking agent (e.g., an antibody orTWEAK-R-Fc) and/or the TNF-α blocking agent is formulated with excipientmaterials, such as sodium chloride, sodium dibasic phosphateheptahydrate, sodium monobasic phosphate, and a stabilizer. It can beprovided, for example, in a buffered solution at a suitableconcentration and can be stored at 2-8° C.

The pharmaceutical compositions may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form can depend on the intended mode of administration andtherapeutic application. Typically compositions for the agents describedherein are in the form of injectable or infusible solutions.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying that yield a powder of an agentdescribed herein plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The proper fluidity of asolution can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prolonged absorption ofinjectable compositions can be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments, the TWEAK blocking agent and/or the TNF-αblocking agent may be prepared with a carrier that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978.

A TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein)can be modified, e.g., with a moiety that improves its stabilizationand/or retention in circulation, e.g., in blood, serum, or othertissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. The modifiedblocking agent can be evaluated to assess whether it can reach sites ofinflammation, e.g., joints.

For example, the TWEAK blocking agent (e.g., an antibody or solubleTWEAK-R protein) can be associated with (e.g., conjugated to) a polymer,e.g., a substantially non-antigenic polymer, such as a polyalkyleneoxide or a polyethylene oxide. Suitable polymers will vary substantiallyby weight. Polymers having molecular number average weights ranging fromabout 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and2,000 to about 12,500) can be used.

For example, a TWEAK blocking agent or TNF-α blocking agent can beconjugated to a water soluble polymer, e.g., a hydrophilic polyvinylpolymer, e.g., polyvinylalcohol or polyvinylpyrrolidone. A non-limitinglist of such polymers includes polyalkylene oxide homopolymers such aspolyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenatedpolyols, copolymers thereof and block copolymers thereof, provided thatthe water solubility of the block copolymers is maintained. Additionaluseful polymers include polyoxyalkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethylene andpolyoxypropylene; polymethacrylates; carbomers; and branched orunbranched polysaccharides.

When the TWEAK blocking agent (e.g., an antibody or soluble TWEAK-Rprotein) is used in combination with a second agent (e.g., a TNF-αblocking agent or other agent described herein), the two agents can beformulated separately or together. For example, the respectivepharmaceutical compositions can be mixed, e.g., just prior toadministration, and administered together or can be administeredseparately, e.g., at the same or different times.

Other therapeutic agents described herein can also be provided aspharmaceutical composition, e.g., by standard methods or methoddescribed herein.

Administration

The TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein)and a TNF-α blocking agent can be administered to a subject, e.g., ahuman subject, by a variety of methods. For many applications, the routeof administration is one of: intravenous injection or infusion (IV),subcutaneous injection (SC), intraperitoneally (IP), or intramuscularinjection. It is also possible to use intra-articular delivery. In somecases, administration may be directly to a site of inflammation, e.g., ajoint or other inflamed site. The blocking agent can be administered asa fixed dose, or in a mg/kg dose.

The dose can also be chosen to reduce or avoid production of antibodiesagainst the TWEAK blocking agent.

The route and/or mode of administration of the blocking agent can alsobe tailored for the individual case, e.g., by monitoring the subject,e.g., using tomographic imaging, neurological exam, and standardparameters associated with the particular disorder, e.g., criteria forassessing rheumatoid arthritis.

Dosage regimens are adjusted to provide the desired response, e.g., atherapeutic response or a combinatorial therapeutic effect. Generally,any combination of doses (either separate or co-formulated) of the TWEAKblocking agent (e.g., an antibody) (and optionally a second agent, e.g.,a TNF-α blocking agent) can be used in order to provide a subject withthe agent in bioavailable quantities. For example, doses in the range of0.1-100 mg/kg, 0.5-100 mg/kg, 1 mg/kg-100 mg/kg, 0.5-20 mg/kg, or 1-10mg/kg can be administered. Other doses can also be used.

Dosage unit form or “fixed dose” as used herein refers to physicallydiscrete units suited as unitary dosages for the subjects to be treated;each unit contains a predetermined quantity of active compoundcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier and optionally in association withthe other agent. Single or multiple dosages may be given. Alternatively,or in addition, the blocking agent may be administered via continuousinfusion.

The TWEAK blocking agent can be administered, e.g., once or twice daily,or about one to four times per week, or preferably weekly, biweekly, ormonthly, e.g., for between about 1 to 10 weeks, preferably between 2 to8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, formulation, route of delivery,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a compound can include a singletreatment or, preferably, can include a series of treatments. Animalmodels can also be used to determine a useful dose, e.g., an initialdose or a regimen.

If a subject is at risk for developing an inflammatory disorder or otherdisorder described herein, the blocking agent can be administered beforethe full onset of the disorder, e.g., as a preventative measure. Theduration of such preventative treatment can be a single dosage of theblocking agent or the treatment may continue (e.g., multiple dosages).For example, a subject at risk for the disorder or who has apredisposition for the disorder may be treated with the blocking agentfor days, weeks, months, or even years so as to prevent the disorderfrom occurring or fulminating.

A pharmaceutical composition may include a “therapeutically effectiveamount” of an agent described herein. Such effective amounts can bedetermined based on the effect of the administered agent, or thecombinatorial effect of agents if more than one agent is used. Atherapeutically effective amount of an agent may also vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the compound to elicit a desired responsein the individual, e.g., amelioration of at least one disorder parameteror amelioration of at least one symptom of the disorder. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

Devices and Kits for Therapy

Pharmaceutical compositions that include the TWEAK blocking agent (e.g.,an antibody or soluble TWEAK-R) can be administered with a medicaldevice. The device can designed with features such as portability, roomtemperature storage, and ease of use so that it can be used in emergencysituations, e.g., by an untrained subject or by emergency personnel inthe field, removed to medical facilities and other medical equipment.The device can include, e.g., one or more housings for storingpharmaceutical preparations that include TWEAK blocking agent, and canbe configured to deliver one or more unit doses of the blocking agent.The device can be further configured to administer a second agent, e.g.,a TNF-α blocking agent, either as a single pharmaceutical compositionthat also includes the TWEAK blocking agent or as two separatepharmaceutical compositions.

For example, the pharmaceutical composition can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;4,790,824; or 4,596,556. Examples of well-known implants and modulesinclude: U.S. Pat. No. 4,487,603, which discloses an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which discloses a therapeutic device foradministering agents through the skin; U.S. Pat. No. 4,447,233, whichdiscloses a medication infusion pump for delivering medication at aprecise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many otherdevices, implants, delivery systems, and modules are also known.

A TWEAK blocking agent (e.g., an antibody or soluble TWEAK-R protein)can be provided in a kit. In one embodiment, the kit includes (a) acontainer that contains a composition that includes a TWEAK blockingagent, and optionally (b) informational material. The informationalmaterial can be descriptive, instructional, marketing or other materialthat relates to the methods described herein and/or the use of theagents for therapeutic benefit.

In an embodiment, the kit also includes a second agent for treating aninflammatory disorder, e.g., a TNF-α blocking agent. For example, thekit includes a first container that contains a composition that includesthe TWEAK blocking agent, and a second container that includes thesecond agent.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods of administering the TWEAK blocking agent (e.g., an antibody orsoluble TWEAK-R protein) and/or TNF-α blocking agent, e.g., in asuitable dose, dosage form, or mode of administration (e.g., a dose,dosage form, or mode of administration described herein), to treat asubject who has had or who is at risk for an inflammatory disorder, orother disorder described herein. The information can be provided in avariety of formats, include printed text, computer readable material,video recording, or audio recording, or information that provides a linkor address to substantive material.

In addition to the blocking agent, the composition in the kit caninclude other ingredients, such as a solvent or buffer, a stabilizer, ora preservative. The blocking agent can be provided in any form, e.g.,liquid, dried or lyophilized form, preferably substantially pure and/orsterile. When the agents are provided in a liquid solution, the liquidsolution preferably is an aqueous solution. When the agents are providedas a dried form, reconstitution generally is by the addition of asuitable solvent. The solvent, e.g., sterile water or buffer, canoptionally be provided in the kit.

The kit can include one or more containers for the composition orcompositions containing the agents. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theagents. The containers can include a combination unit dosage, e.g., aunit that includes both the TWEAK blocking agent and the second agent,e.g., in a desired ratio. For example, the kit includes a plurality ofsyringes, ampules, foil packets, blister packs, or medical devices,e.g., each containing a single combination unit dose. The containers ofthe kits can be air tight, waterproof (e.g., impermeable to changes inmoisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe or other suitable delivery device. Thedevice can be provided pre-loaded with one or both of the agents or canbe empty, but suitable for loading.

Nucleic Acid and Protein Analysis

Numerous methods for detecting TWEAK or TWEAK-R protein and nucleic acidas well as proteins and nucleic acids for other biomarkers describedherein (including those listed in Table 1) are available to the skilledartisan, including antibody-based methods for protein detection (e.g.,Western blot or ELISA), and hybridization-based methods for nucleic aciddetection (e.g., PCR or Northern blot).

Arrays are particularly useful molecular tools for characterizing asample, e.g., a sample from a subject. For example, an array havingcapture probes for multiple genes, including probes for TWEAK and/orother biomarkers, or for multiple proteins, can be used in a methoddescribed herein. Altered expression of TWEAK (or other biomarkerprovided herein) nucleic acids and/or protein can be used to evaluate asample, e.g., a sample from a subject, e.g., to evaluate a disorderdescribed herein.

Arrays can have many addresses, e.g., locatable sites, on a substrate.The featured arrays can be configured in a variety of formats,non-limiting examples of which are described below. The substrate can beopaque, translucent, or transparent. The addresses can be distributed,on the substrate in one dimension, e.g., a linear array; in twodimensions, e.g., a planar array; or in three dimensions, e.g., a threedimensional array. The solid substrate may be of any convenient shape orform, e.g., square, rectangular, ovoid, or circular.

Arrays can be fabricated by a variety of methods, e.g.,photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;5,510,270; and 5,527,681), mechanical methods (e.g., directed-flowmethods as described in U.S. Pat. No. 5,384,261), pin based methods(e.g., as described in U.S. Pat. No. 5,288,514), and bead basedtechniques (e.g., as described in PCT US/93/04145).

The capture probe can be a single-stranded nucleic acid, adouble-stranded nucleic acid (e.g., which is denatured prior to orduring hybridization), or a nucleic acid having a single-stranded regionand a double-stranded region. Preferably, the capture probe issingle-stranded. The capture probe can be selected by a variety ofcriteria, and preferably is designed by a computer program withoptimization parameters. The capture probe can be selected to hybridizeto a sequence rich (e.g., non-homopolymeric) region of the gene. TheT_(m) of the capture probe can be optimized by prudent selection of thecomplementarity region and length. Ideally, the T_(m) of all captureprobes on the array is similar, e.g., within 20, 10, 5, 3, or 2° C. ofone another.

The isolated nucleic acid is preferably mRNA that can be isolated byroutine methods, e.g., including DNase treatment to remove genomic DNAand hybridization to an oligo-dT coupled solid substrate (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y). The substrate is washed, and the mRNA is eluted.

The isolated mRNA can be reversed transcribed and optionally amplified,e.g., by rtPCR, e.g., as described in (U.S. Pat. No. 4,683,202). Thenucleic acid can be an amplification product, e.g., from PCR (U.S. Pat.Nos. 4,683,196 and 4,683,202); rolling circle amplification (“RCA,” U.S.Pat. No. 5,714,320), isothermal RNA amplification or NASBA (U.S. Pat.Nos. 5,130,238; 5,409,818; and 5,554,517), and strand displacementamplification (U.S. Pat. No. 5,455,166). The nucleic acid can be labeledduring amplification, e.g., by the incorporation of a labelednucleotide. Examples of preferred labels include fluorescent labels,e.g., red-fluorescent dye Cy5 (Amersham) or green-fluorescent dye Cy3(Amersham), and chemiluminescent labels, e.g., as described in U.S. Pat.No. 4,277,437. Alternatively, the nucleic acid can be labeled withbiotin, and detected after hybridization with labeled streptavidin,e.g., streptavidin-phycoerythrin (Molecular Probes).

The labeled nucleic acid can be contacted to the array. In addition, acontrol nucleic acid or a reference nucleic acid can be contacted to thesame array. The control nucleic acid or reference nucleic acid can belabeled with a label other than the sample nucleic acid, e.g., one witha different emission maximum. Labeled nucleic acids can be contacted toan array under hybridization conditions. The array can be washed, andthen imaged to detect fluorescence at each address of the array.

The expression level of a TWEAK or other biomarker can be determinedusing an antibody specific for the polypeptide (e.g., using a westernblot or an ELISA assay). Moreover, the expression levels of multipleproteins, including TWEAK and the exemplary biomarkers provided herein,can be rapidly determined in parallel using a polypeptide array havingantibody capture probes for each of the polypeptides. Antibodiesspecific for a polypeptide can be generated by a method described herein(see “Antibody Generation”). The expression level of a TWEAK and theexemplary biomarkers provided herein can be measured in a subject (e.g.,in vivo imaging) or in a biological sample from a subject (e.g., blood,serum, plasma, or synovial fluid).

A low-density (96 well format) protein array has been developed in whichproteins are spotted onto a nitrocellulose membrane (Ge (2000) NucleicAcids Res. 28, e3, I-VII). A high-density protein array (100,000 sampleswithin 222×222 mm) used for antibody screening was formed by spottingproteins onto polyvinylidene difluoride (PVDF) (Lueking et al. (1999)Anal. Biochem. 270:103-111). See also, e.g., Mendoza et al. (1999).Biotechniques 27:778-788; MacBeath and Schreiber (2000) Science289:1760-1763; and De Wildt et al. (2000) Nature Biotech. 18:989-994.These art-known methods and other can be used to generate an array ofantibodies for detecting the abundance of polypeptides in a sample. Thesample can be labeled, e.g., biotinylated, for subsequent detection withstreptavidin coupled to a fluorescent label. The array can then bescanned to measure binding at each address.

The nucleic acid and polypeptide arrays of the invention can be used ina wide variety of applications. For example, the arrays can be used toanalyze a patient sample. The sample is compared to data obtainedpreviously, e.g., known clinical specimens or other patient samples.Further, the arrays can be used to characterize a cell culture sample,e.g., to determine a cellular state after varying a parameter, e.g.,exposing the cell culture to an antigen, a transgene, or a testcompound.

The expression data can be stored in a database, e.g., a relationaldatabase such as a SQL database (e.g., Oracle or Sybase databaseenvironments). The database can have multiple tables. For example, rawexpression data can be stored in one table, wherein each columncorresponds to a gene being assayed, e.g., an address or an array, andeach row corresponds to a sample. A separate table can store identifiersand sample information, e.g., the batch number of the array used, date,and other quality control information.

Expression profiles obtained from gene expression analysis on an arraycan be used to compare samples and/or cells in a variety of states asdescribed in Golub et al. ((1999) Science 286:531). In one embodiment,expression (e.g., mRNA expression or protein expression) information fora gene encoding TWEAK and/or a gene encoding a exemplary biomarkerprovided herein are evaluated, e.g., by comparison a reference value,e.g., a reference value. Reference values can be obtained from acontrol, e.g., a reference subject. Reference values can also beobtained from statistical analysis, e.g., to provide a reference valuefor a cohort of subject, e.g., age and gender matched subject, e.g.,normal subjects or subject who have rheumatoid arthritis or otherdisorder described herein. Statistical similarity to a particularreference (e.g., to a reference for a risk-associated cohort) or anormal cohort can be used to provide an assessment (e.g., an indicationof risk of inflammatory disorder) to a subject, e.g., a subject who hasnot been diagnosed with a disorder described herein.

Subjects suitable for treatment can also be evaluated for expressionand/or activity of TWEAK and/or other biomarker. Subjects can beidentified as suitable for treatment (e.g., with a TWEAK blockingagent), if the expression and/or activity for TWEAK and/or the otherbiomarker is elevated relative to a reference, e.g., reference value,e.g., a reference value associated with normal.

Subjects who are being administered an agent described herein or othertreatment can be evaluated as described for expression and/or activityof TWEAK and/or other biomarkers described herein. The subject can beevaluated at multiple times. e.g., at multiple times during a course oftherapy, e.g., during a therapeutic regimen. Treatment of the subjectcan be modified depending on how the subject is responding to thetherapy. For example, a reduction in TWEAK expression or activity or areduction in the expression or activity of genes stimulated by TWEAK canbe indicative of responsiveness.

Particular effects mediated by an agent may show a difference (e.g.,relative to an untreated subject, control subject, or other reference)that is statistically significant (e.g., P value <0.05 or 0.02).Statistical significance can be determined by any art known method.Exemplary statistical tests include: the Students T-test, Mann Whitney Unon-parametric test, and Wilcoxon non-parametric statistical test. Somestatistically significant relationships have a P value of less than 0.05or 0.02.

Methods of Evaluating Genetic Material

There are numerous methods for evaluating genetic material to providegenetic information. These methods can be used to evaluate a geneticlocus that includes a gene encoding TWEAK or a gene encoding a biomarkerdescribed herein. The methods can be used to evaluate one or morenucleotides, e.g., a coding or non-coding region of the gene, e.g., in aregulatory region (e.g., a promoter, a region encoding an untranslatedregion or intron, and so forth).

Nucleic acid samples can analyzed using biophysical techniques (e.g.,hybridization, electrophoresis, and so forth), sequencing, enzyme-basedtechniques, and combinations-thereof. For example, hybridization ofsample nucleic acids to nucleic acid microarrays can be used to evaluatesequences in an mRNA population and to evaluate genetic polymorphisms.Other hybridization based techniques include sequence specific primerbinding (e.g., PCR or LCR); Southern analysis of DNA, e.g., genomic DNA;Northern analysis of RNA, e.g., mRNA; fluorescent probe based techniques(see, e.g., Beaudet et al. (2001) Genome Res. 11(4):600-8); and allelespecific amplification. Enzymatic techniques include restriction enzymedigestion; sequencing; and single base extension (SBE). These and othertechniques are well known to those skilled in the art.

Electrophoretic techniques include capillary electrophoresis andSingle-Strand Conformation Polymorphism (SSCP) detection (see, e.g.,Myers et al. (1985) Nature 313:495-8 and Ganguly (2002) Hum Mutat.19(4):334-42). Other biophysical methods include denaturing highpressure liquid chromatography (DHPLC).

In one embodiment, allele specific amplification technology that dependson selective PCR amplification may be used to obtain geneticinformation. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucl. Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it is possible to introduce a restriction site in the regionof the mutation to create cleavage-based detection (Gasparini et al.(1992) Mol. Cell. Probes 6:1). In another embodiment, amplification canbe performed using Taq ligase for amplification (Barany (1991) Proc.Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only ifthere is a perfect match at the 3′ end of the 5′ sequence making itpossible to detect the presence of a known mutation at a specific siteby looking for the presence or absence of amplification.

Enzymatic methods for detecting sequences include amplificationbased-methods such as the polymerase chain reaction (PCR; Saiki, et al.(1985) Science 230:1350-1354) and ligase chain reaction (LCR; Wu. et al.(1989) Genomics 4:560-569; Barringer et al. (1990), Gene 1989:117-122;F. Barany (1991) Proc. Natl. Acad. Sci. USA 1988:189-193);transcription-based methods utilize RNA synthesis by RNA polymerases toamplify nucleic acid (U.S. Pat. Nos. 6,066,457; 6,132,997; and5,716,785; Sarkar et al., (1989) Science 244:331-34; Stofler et al.,(1988) Science 239:491); NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and5,554,517); rolling circle amplification (RCA; U.S. Pat. Nos. 5,854,033and 6,143,495) and strand displacement amplification (SDA; U.S. Pat.Nos. 5,455,166 and 5,624,825). Amplification methods can be used incombination with other techniques.

Other enzymatic techniques include sequencing using polymerases, e.g.,DNA polymerases and variations thereof such as single base extensiontechnology. See, e.g., U.S. Pat. Nos. 6,294,336; 6,013,431; and5,952,174.

Fluorescence based detection can also be used to detect nucleic acidpolymorphisms. For example, different terminator ddNTPs can be labeledwith different fluorescent dyes. A primer can be annealed near orimmediately adjacent to a polymorphism, and the nucleotide at thepolymorphic site can be detected by the type (e.g., “color”) of thefluorescent dye that is incorporated.

Hybridization to microarrays can also be used to detect polymorphisms,including SNPs. For example, a set of different oligonucleotides, withthe polymorphic nucleotide at varying positions with theoligonucleotides can be positioned on a nucleic acid array. The extentof hybridization as a function of position and hybridization tooligonucleotides specific for the other allele can be used to determinewhether a particular polymorphism is present. See, e.g., U.S. Pat. No.6,066,454.

In one implementation, hybridization probes can include one or moreadditional mismatches to destabilize duplex formation and sensitize theassay. The mismatch may be directly adjacent to the query position, orwithin 10, 7, 5, 4, 3, or 2 nucleotides of the query position.Hybridization probes can also be selected to have a particular T_(m),e.g., between 45-60° C., 55-65° C., or 60-75° C. In a multiplex assay,T_(m)'s can be selected to be within 5, 3, or 2° C. of each other.

It is also possible to directly sequence the nucleic acid for aparticular genetic locus, e.g., by amplification and sequencing, oramplification, cloning and sequence. High throughput automated (e.g.,capillary or microchip based) sequencing apparati can be used. In stillother embodiments, the sequence of a protein of interest is analyzed toinfer its genetic sequence. Methods of analyzing a protein sequenceinclude protein sequencing, mass spectroscopy, sequence/epitope specificimmunoglobulins, and protease digestion.

Any combination of the above methods can also be used. The above methodscan be used to evaluate any genetic locus, e.g., in a method foranalyzing genetic information from particular groups of individuals orin a method for analyzing a polymorphism associated with a disorderdescribed herein, e.g., rheumatoid arthritis, e.g., in a gene encodingTWEAK or another biomarker described herein.

EXAMPLES Example 1 Exemplary Sequences

An exemplary sequence of a human TWEAK protein is as follows

(SEQ ID NO: 1) MAARRSQRRR GRRGEPGTAL LVPLALGLGL ALACLGLLLA VVSLGSRASLSAQEPAQEEL VAEEDQDPSE LNPQTEESQD PAPFLNRLVR PRRSAPKGRK TRARRAIAAHYEVHPRPGQD GAQAGVDGTV SGWEEARINS SSPLRYNRQI GEFIVTRAGL YYLYCQVHFDEGKAVYLKLD LLVDGVLALR CLEEFSATAA SSLGPQLRLC QVSGLLALRP GSSLRIRTLPWAHLKAAPFL TYFGLFQVH

An exemplary sequence of a human Fn14 protein is as follows:

(SEQ ID NO: 2) MARGSLRRLL RLLVLGLWLA LLRSVAGEQA PGTAPCSRGS SWSADLDKCMDCASCRARPH SDFCLGCAAA PPAPFRLLWP ILGGALSLTF VLGLLSGFLV WRRCRRREKFTTPIEETGGE GCPAVALIQ

Example 2 Genes that are Synergistically Activated by TWEAK and TNF-α

Microarrays were analyzed to identify genes whose expression in humansynoviocytes was induced by TWEAK and TNF-α. The following are examplesof genes that are synergistically activated by TWEAK and TNF-α.

TABLE 1 Genes Synergistically Activated by TWEAK and TNF-α AffyIDannotation 208229_at — 216064_s_at — 220396_at — 222332_at — 207999_s_atadenosine deaminase, RNA-specific, B1 (RED1 homolog rat) 202109_atADP-ribosylation factor interacting protein 2 (arfaptin 2) 201444_s_atATPase, H+ transporting, lysosomal accessory protein 2 210538_s_atbaculoviral IAP repeat-containing 3 221534_at basophilic leukemiaexpressed protein BLES03 203773_x_at biliverdin reductase A 205733_atBloom syndrome 211314_at calcium channel, voltage-dependent, alpha 1Gsubunit 217118_s_at chromosome 22 open reading frame 9 216607_s_atcytochrome P450, family 51, subfamily A, polypeptide 1 213279_atdehydrogenase/reductase (SDR family) member 1 209703_x_at DKFZP586A0522protein 210839_s_at ectonucleotide pyrophosphatase/phosphodiesterase 2(autotaxin) 210002_at GATA binding protein 6 212241_at glutamatereceptor, ionotropic, N-methyl D-aspartate-like 1A 208055_s_at hectdomain and RLD 4 204512_at human immunodeficiency virus type I enhancerbinding protein 1 216510_x_at immunoglobulin heavy constant gamma 1 (G1mmarker) 201548_s_at Jumonji, AT rich interactive domain 1B (RBP2-like)220972_s_at keratin associated protein 9-9 212805_at KIAA0367212546_s_at KIAA0826 215680_at KIAA1654 protein 218906_x_at likelyortholog of kinesin light chain 2 210104_at mediator of RNA polymeraseII transcription, subunit 6 homolog (yeast) 214397_at methyl-CpG bindingdomain protein 2 212713_at microfibrillar-associated protein 4 203901_atmitogen-activated protein kinase kinase kinase 7 interacting protein 1213040_s_at neuronal pentraxin receptor 202783_at nicotinamidenucleotide transhydrogenase 211691_x_at Ornithine decarboxylase antizyme4 mRNA, complete cds 205991_s_at paired related homeobox 1 204715_atpannexin 1 214735_at phosphoinositide-binding protein PIP3-E 203709_atphosphorylase kinase, gamma 2 (testis) 207709_at protein kinase,AMP-activated, alpha 2 catalytic subunit 213136_at protein tyrosinephosphatase, non-receptor type 2 213524_s_at putative lymphocyte G0/G1switch gene 202388_at regulator of G-protein signalling 2, 24 kDa218441_s_at RNA polymerase II associated protein 1 212140_at SCC-112protein 201471_s_at sequestosome 1 212609_s_at serologically definedcolon cancer antigen 8 212393_at SET binding factor 1 214931_s_at SFRSprotein kinase 2 M97935_MB_at signal transducer and activator oftranscription 1, 91 kDa 204804_at Sjogren syndrome antigen A1 (52 kDa,ribonucleoprotein autoantigen SS-A/Ro) 214925_s_at spectrin, alpha,non-erythrocytic 1 (alpha-fodrin) 221268_s_at sphingosine-1-phosphatephosphatase 1 212154_at syndecan 2 (heparan sulfate proteoglycan 1, cellsurface-associated, fibroglycan) 212800_at syntaxin 6 201449_at TIA1cytotoxic granule-associated RNA binding protein 216241_s_attranscription elongation factor A (SII), 1 201399_s_at translocationassociated membrane protein 1 210372_s_at tumor protein D52-like 1206959_s_at UPF3 regulator of nonsense transcripts homolog A (yeast)219393_s_at v-akt murine thymoma viral oncogene homolog 3 (proteinkinase B, gamma) 205205_at v-rel reticuloendotheliosis viral oncogenehomolog B, nuclear factor of kappa light polypeptide gene enhancer inB-cells 3 (avian) Probe Set Id Gene Title 1405_i_at Chemokine (C-Cmotif) ligand 5 (CCL5) 204490_s_at CD44 antigen (homing function andIndian blood group system) (CD44) 204655_at RANTES (SCYA5) 205619_s_aymesenchyme homeo box 1 (MEOX1) platelet-derived growth factorreceptor-like . . . (NM_006207) fibroblast growth factor receptor 4fibroblast growth factor 22 chemokine (C-C motif) ligand 18

Still other genes are activated by both (i) TWEAK in the absence ofTNF-α and (ii) TNF-α in the absence of TWEAK.

Example 3 Effect of Combination of Blocking TWEAK and TNF in mCIA asMeasured by an Average Arthritis Index

The mouse collagen-induced arthritis (mCIA) model is a commonly-usedmodel (see e.g., Stuart et al., J. Clin. Invest. 69:673-683 (1982)) ofrheumatoid arthritis. A mCIA model was used to study the effects of acombination anti-TWEAK and anti-TNF-α treatment on arthritisdevelopment. Arthritis was induced in mice via collagen immunization(CII/CFA: collagen II and complete Freud's adjuvant). Anti-TWEAKmonoclonal antibody (mu anti-TWEAK mAb+hu IgG1); soluble TNF-α receptor(TNFr-hu Fc+mu IgG2a); a combination of anti-TWEAK monoclonal antibodyand soluble TNF-α receptor (mu anti-TWEAK mAb+TNFr-hu Fc); or PBS orisotype-matched negative controls were administered on days 20, 23, 27,30, and 34 after collagen immunization. Each treatment group containedten mice. Each antibody was administered at a dose of 10 mg/kg.Arthritis was assessed using an average arthritis index (see e.g., Li etal., Arthritis Res. Ther. 6:R273—R281 (2004)). Four paws were measuredper mouse using the scoring system: 0=normal paw; 1=swelling ofindividual digits; 2=moderate swelling and redness of ankle or wristjoints; 3=swelling and redness of at least two joints; and 4=swelling ofthe whole paw. The sum of the four paw scores (y axis) were plottedagainst the days after collagen immunization (x axis).

FIG. 1 shows the results of this study. The mice treated with thecombination of anti-TWEAK and anti-TNF-α agents had a lower averagearthritis index score than mice treated with either blocking agent aloneor the controls. Also, as indicated on the right side of the graph under“% incidence,” the mice treated with the combination had a lower overallincidence of arthritis (60%) than mice treated with either agent alone(67% for anti-TNF-α treatment; 80% for anti-TWEAK mAb treatment) or withthe controls (80% for PBS treatment; 90% for isotype matched antibodytreatment).

Example 4 Effect of Combination of Blocking TWEAK and TNF in mCIA asMeasured by Average Metatarsal Height

The mCIA model was used to study the effects of a combination anti-TWEAKand anti-TNF-α therapy on arthritis development as measured by averagemetatarsal height/paw thickness (see e.g., Campo et al., Arthritis Res.Ther. 5:R122-R131 (2003)). Mice were treated with anti-TWEAK monoclonalantibody (mu anti-TWEAK mAb+hu IgG1); soluble TNF-α receptor (TNFRp55:huFc+mu IgG2a); a combination of anti-TWEAK monoclonal antibody andsoluble TNF-α receptor (mu anti-TWEAK mAb+TNFRp55:hu Fc); or PBS orisotype-matched negative controls on days 20, 23, 27, 30, and 34 aftercollagen immunization. Each treatment group contained ten mice. Eachantibody was administered at a dose of 10 mg/kg. Metatarsal height wasmeasured using calipers 38 days after collagen immunization. The averagemetatarsal height (y axis) for each mouse per treatment (x axis) wasplotted.

FIG. 2 shows the results of this study. Mice treated with thecombination of anti-TWEAK and anti-TNF-α agents hadstatistically-significant lower average metatarsal height values thanmice treated with either blocking agent alone or the controls (*p<0.05for the average value per treatment when compared to the controls).

Example 5 Effect of Combination of Blocking TWEAK and TNF in mCIA asMeasured by Body Weight Change

The mCIA model was used to study the effects of a combination anti-TWEAKand anti-TNF-α therapy on arthritis development as measured by percentbody weight change (Campo et al., Arthritis Res. Ther. 5:R122-R131(2003)). Mice were treated with anti-TWEAK monoclonal antibody (muanti-TWEAK mAb+hu IgG1 (an isotype matched control for TNFRp55:hu Fc));soluble TNF-α receptor (TNFRp55:hu Fc+mu IgG2a); a combination ofanti-TWEAK monoclonal antibody and soluble TNF-α receptor (mu anti-TWEAKmAb+TNFRp55:hu Fc); or PBS or isotype-matched negative controls on days20, 23, 27, 30, and 34 after collagen immunization. Each treatment groupcontained ten mice. Each antibody was administered at a dose of 10mg/kg. Mice were weighed at various time points after collagenimmunization and the percent change in body weight were calculated pertreatment. The percent body weight change for each treatment (y axis)was plotted against the days after arthritis induction by collagenimmunization (x axis).

FIG. 3 shows the results of this study. Mice treated with thecombination of anti-TWEAK and anti-TNF-α agents had astatistically-significant smaller percent change in body weight thanmice treated with either blocking agent alone or the controls (*p<0.01for the value per treatment when compared to the controls or to theTNFRp55:hu Fc+mu IgG2a treated mice).

Example 6 TWEAK Induced Genes

We applied TWEAK doses (5 ng/ml and 50 ng/ml) to cells at both 6 and 24hour time points, and observed that some genes are modulated by TWEAKonly. These genes were not affected by application of TNF-α, even at aconcentration of 5 ng/ml. Examples of such genes are:

TABLE 2 1. NIK/mitogen-activated protein kinase kinase kinase14(MAP3K14) 2. Homo sapiens cDNA FLJ11796 fis, clone HEMBA1006158,highly similar to Homo sapiens transcription factor forkhead-like 7(FKHL7) gene 3. similar to glucosamine-6-sulfatases Homo sapiens serumglucocorticoid regulated kinase (SGK), mRNA 4. Homo sapiens REV3 (yeasthomolog)-like, catalytic subunit of DNA polymerase zeta (REV3L), mRNA.5. ADAM 10/a disintegrin and metalloproteinase domain 10 6. nuclearfactor (erythroid-derived 2)-like 1 7. Homo sapiens SerArg-relatednuclear matrix protein (plenty of prolines 101-like) (SRM160), mRNA. 8.Homo sapiens doublecortin and CaM kinase-like 1 (DCAMKL1), mRNA. 9. Homosapiens Cdc42 effector protein 4; binder of Rho GTPases 4 (CEP4), mRNA.10. Homo sapiens mRNA; cDNA DKFZp762L106 (from clone DKFZp762L106);partial cds. In addition, in normal human synoviocytes, CBR3 and IL8 areinduced by TWEAK treatment (5 ng/ml) alone.

Example 7 Experiments with TWEAK

FIG. 4 shows that treatment with TWEAK-blocking monoclonal antibodiescan lessen the development of arthritis in both mouse and rat CIA modelsof arthritis. The left panel shows that treatment with an anti-TWEAKantibody (murine anti-TWEAK mAb) results in a lower value in the averagearthritis index, as compared to treatment with a control antibody(mIgG2a), in a mouse CIA model in which arthritis was induced withCII/CFA. The right panel shows that treatment with an anti-TWEAKantibody (anti-TWEAK mAb) results in a lower value in the averagearthritis index, as compared to treatment with a control antibody (Ha4/8) or PBS in a rat CIA model, in which arthritis was induced withcollagen II and incomplete Freud's adjuvant (CII/IFA).

FIG. 5 shows that TWEAK-blocking monoclonal antibodies can beadministered at the same time as (Dosing scheme 1) or after (Dosingscheme 2) the induction of arthritis by collagen immunization and stillhave the effect of lessening the development of arthritis in both mouseand rat CIA models of arthritis. The left panel shows that an anti-TWEAKantibody can be administered prior to or after the induction ofarthritis to effect a lower value in the average arthritis index, ascompared to administration of a control antibody (mIgG2a), in a mouseCIA model. The right panel shows that an anti-TWEAK antibody can beadministered prior to or after the induction of arthritis to effect alower value in the average arthritis index, as compared toadministration of a control antibody (Ha 4/8) or PBS, in a rat CIAmodel.

FIG. 6 shows that anti-TWEAK monoclonal antibody (ABG. 11) treatmentdecreases inflammation in the rat CIA model, as measured by a clinicalpaw score and an overall inflammation score; the treatment alsodecreases cartilage and bone loss, as measured by the parameters of boneabsorption, decrease in toluidine blue uptake, and loss of articularcartilage. Similar results were seen in the mouse CIA model.

FIG. 7 shows serum TWEAK levels in the mouse CIA model at various timepoints (day (D) 23, 28, 30, and 38) after induction of arthritis. TWEAKlevels were elevated as compared to the levels in control mice (DBA/1).

FIG. 8 shows the levels of MMP9, lymphotactin, IP— 10, and IL-6 atvarious time points (day 23, 30, and 40) after induction of arthritis inthe mouse CIA model. Treatment with anti-TWEAK monoclonal antibody (P5G9and P5G9 (Full, also termed dosing scheme 1)) prevented as great anincrease in the levels of these proteins, as compared to the levels inmice treated with a control (mIgG2a) or in mice not immunized to developarthritis (normal DBA). Similar results were seen in the rat CIA model.

Experiments were performed to demonstrate that inhibition of TWEAK withanti-TWEAK antibodies does not affect the adaptive immune response.After collagen immunization, mice that had been treated with anti-TWEAKmonoclonal antibodies were able to mount collagen-specific B cell and Tcell responses to a similar extent as mice that had been treated with acontrol, isotype-matched antibody (mIgG2a; data not shown).

Experiments were performed to measure the levels of Fn14 (TWEAKreceptor) on primary human cell types found in a joint:fibroblast-likesynoviocytes, articular chondrocytes, and osteoblasts.Fluorescence-activated cell sorting experiments using anti-Fn14 antibody(ITEM-4) or a control antibody (anti-mFc) demonstrated that Fn14 levelswere elevated above background in all three cells types, with levelsbeing higher in the synoviocytes and osteoblasts than in thechondrocytes.

Experiments were performed to demonstrate that TWEAK and TNF-α can eachstimulate matrix metalloprotease production by chondrocytes. MMP-1,MMP-2, MMP-3, and MMP-9 levels all increased after treatment with TWEAK(100 ng/ml) or TNF-α (50 ng/ml).

Experiments were performed to demonstrate the agonistic, synergisticeffects of TWEAK and TNF-α. Human fibroblast-like synoviocytes weretreated with varying concentrations of TWEAK alone, TNF-α alone, or acombination of TWEAK and TNF-α, and the level of RANTES production witheach treatment was measured by ELISA. Both TWEAK and TNF-α inducedRANTES production. However, when TWEAK and TNF-α were administered incombination, a synergistic level of RANTES production resulted. Thus,TWEAK and TNF-α can synergize to induce expression of particularinflammatory genes.

Example 8 Genes Induced by TWEAK and TNF-α Combination Treatment inNormal Synoviocytes

Synoviocytes from a healthy donor were cultured in vitro and treatedwith 5 ng/ml TWEAK and 0.5 ng/ml TNF-α. Table 3 lists genes whoseexpression was affected by the treatment with TWEAK and TNF-α to astatistically significant degree. The genes are grouped by their geneontology category.

TABLE 3 Go Ontology Protein P Value Positive regulation of CASP1, CFLAR,LGLAS9, Myd88, 5.81e−018 IκB SECTM1, TNFSF10, TRIM38 Inflammatory CCL3,CCL4, CCL7, CCL8, 9.94e−007 Response CXCL9, ILRN, Myd88, TLR3 ChemotaxisCCL3, CCL4, CCL7, CCL8, 0.0003 CXCL9, ERG1, SOCS1 Interferon ResponseIFI44, WARS, IRF2 0.001The changes were identified as statistically significant Go categoriesbased on hypergeometric mean.

Example 9 Genes Induced by TWEAK and TNF-α Combination Treatment in RASynoviocytes

Synoviocytes from a rheumatoid arthritis patient donor were cultured invitro and treated with 5 ng/ml TWEAK and 0.5 ng/ml TNF-α. Table 4 listsgenes whose expression was affected by the treatment with TWEAK andTNF-α to a statistically significant degree. The genes are grouped bytheir gene ontology category.

TABLE 4 Go Ontology Protein pValue Inflammatory CXCL10, CXCL3, PTGS2,APOL3 4.26e−005 Response Response to Stress CXCL10, CXCL3, PTGS2, APOL3,5.56e−006 MDA5, MX1, PTGES, Rig-1 Response to biotic CXCL10, CXCL3,PTGS2, APOL3, 3.69e−009 stimuli MDA5, MX1, PTGES, Rig-1, GBp1The changes were identified as statistically significant Go categoriesbased on hypergeometric mean.

Example 10

P2D10 is an exemplary murine anti-TWEAK antibody. The sequence of themurine P2D10 heavy chain variable domain (SEQ ID NO:3), with CDRsunderlined is:

1 EVQLVESGGG LVRPGGSLKL FCAASGFTFS RYAMSWVRQS PEKRLEWVAE 51ISSGGSYPYY PDTVTGRFTI SRDNAKNTLY LEMSSLKSED TAMYYCARVL 101YYDYDGDRIE VMDYWGQGTA VIVSS

This is a murine subgroup 3D heavy chain variable domain.

The sequence of the murine P2D10 light chain variable domain (SEQ IDNO:4), with CDRs underlined is:

1 DVVMTQSPLS LSVSLGDQAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPK 51FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVAAEDLGV YFCSQSTHFP 101 RTFGGGTTLE IK

This is a murine subgroup 2 kappa light chain.

This is an exemplary amino acid sequence of the mature huP2D10 H1 IgG1heavy chain (SEQ ID NO:5):

1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYAMSWVRQA PGKGLEWVAE 51 ISSGGSYPYYPDTVTGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVL 101 YYDYDGDRIE VMDYWGQGTLVTVSSASTKG PSVFPLAPSS KSTSGGTAAL 151 GCLVKDYFPE PVTVSWNSGA LTSGVHTFPAVLQSSGLYSL SSVVTVPSSS 201 LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAPELLGGPSVFL 251 FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR301 EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ 351PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK 401 TTPPVLDSDGSFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS 451 LSPG

This is an exemplary amino acid sequence of the mature huP2D10 L1 lightchain (SEQ ID NO:6):

1 DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPQ 51 FLIYKVSNRFSGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YFCSQSTHFP 101 RTFGGGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK 151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSLSSTLTLSKAD YEKHKVYACE 201 VTHQGLSSPV TKSFNRGEC

This is an exemplary amino acid sequence of the mature huP2D10 L2 lightchain (SEQ ID NO:7):

1 DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPQ 51 LLIYKVSNRFSGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHFP 101 RTFGGGTKVE IKRTVAAPSVFIFPPSDEQL KSGTASVVCL LNNFYPREAK 151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSLSSTLTLSKAD YEKHKVYACE 201 VTHQGLSSPV TKSFNRGEC

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method comprising: administering, to a human subject who hasrheumatoid arthritis, a TWEAK blocking agent in combination with a TNF-αblocking agent, in amounts and for a time to provide a therapeuticeffect.
 2. The method of claim 1 wherein the TWEAK blocking agent andthe TNF-α blocking agent are administered in an amount effective toinhibit TWEAK and TNF-α in synoviocytes, chondrocytes, osteoclasts, orosteoblasts.
 3. The method of claim 1 wherein the TWEAK blocking agentand the TNF-α blocking agent are administered in an amount effective toreduce transcription of a set of genes induced by TWEAK and TNF-αcellular programs in synoviocytes, chondrocytes, osteoclasts, orosteoblasts.
 4. The method of claim 1 wherein the TWEAK blocking agentand the TNF-α blocking agent are administered in an amount effective toreduce transcription of one or more genes list in Table 1 insynoviocytes, chondrocytes, osteoclasts, or osteoblasts.
 5. The methodof claim 1 wherein the TWEAK blocking agent is administered in an amountthat is at least 20% less than standard dosages for TWEAK blocking agentmonotherapy for treating an adult subject for rheumatoid arthritis. 6.The method of claim 1 or 5 wherein the TNF-α blocking agent isadministered in an amount that is at least 20% less than standarddosages for TNF-α blocking agent monotherapy for treating an adultsubject for rheumatoid arthritis.
 7. The method of claim 1 wherein thesubject is not receiving methotrexate.
 8. The method of claim 1 whereinthe subject is not receiving any other disease modifying anti-rheumaticdrug (DMARD).
 9. The method of claim 1 wherein the amounts result in astatistically significant reduction in joint damage as measured by theSharp erosion score.
 10. The method of claim 1 wherein the TWEAKblocking agent reduces the ability of TWEAK to bind to a TWEAK receptor.11. The method of claim 1 wherein the TWEAK blocking agent is anantibody that binds to TWEAK.
 12. The method of claim 1 wherein theTWEAK blocking agent comprises an antibody that binds to a TWEAKreceptor.
 13. The method of claim 1 wherein the TWEAK blocking agentcomprises a soluble form of a TWEAK receptor.
 14. The method of claim 1wherein the TWEAK blocking soluble form of the TWEAK receptor is fusedwith an antibody Fc region.
 15. The method of claim 1 wherein the TNF-αblocking agent reduces the ability of TNF-α to bind to a TNF-α receptor.16. The method of claim 1 wherein the TNF-α blocking agent comprises anantibody that binds to TNF-α, TNFR-I, or TNFR-II.
 17. The method ofclaim 16 wherein the TNF-α blocking agent is infliximab or adalimumab.18. The method of claim 1 wherein the TNF-α blocking agent comprises asoluble form of a TNF-α receptor.
 19. The method of claim 1 wherein theTNF-α blocking agent is etanercept.
 20. The method of claim 1 furthercomprising: evaluating the subject using a total Sharp score (TSS),Sharp erosion score, HAQ disability index, or radiologic method.
 21. Amethod comprising: administering, to a human subject who has rheumatoidarthritis, a TWEAK blocking agent in combination with another biologicDMARD, in amounts and for a time to provide a therapeutic effect.
 22. Akit that comprises a TWEAK blocking agent and a TNF-α blocking agent,wherein the agents are provided as separate pharmaceutical compositionsor a single pharmaceutical composition.
 23. The kit of claim 22 furthercomprising instructions for administration to treat rheumatoidarthritis.
 24. A pharmaceutical composition that includes: a TWEAKblocking agent; and a TNF-α blocking agent.
 25. A method comprising:identifying a subject who has TWEAK and TNF-α mediated inflammation; andadministering to the subject: (i) a TWEAK blocking agent; (ii) a TNF-αblocking agent; or (iii) a combination of (i) and (ii).
 26. The methodof claim 25 wherein the subject is identified by evaluating expressionof one or more genes in synoviocytes from the subject.
 27. The method ofclaim 26 wherein the evaluated one or more genes include one or moregenes selected from Table
 1. 28. The method of claim 26 wherein theevaluating comprises obtaining a sample of cells or tissue from thesubject.
 29. The method of claim 25 wherein the identifying furthercomprises detecting symptoms of rheumatoid arthritis in the subject. 30.A method comprising: administering, to a human subject who hasrheumatoid arthritis, and who is being or has been withdrawn from aTNF-α blocking agent, a TWEAK blocking agent in an amount and for a timeeffective to provide a therapeutic effect.
 31. The method of claim 30wherein the TNF-α blocking agent causes toxicity, induces animmune-compromised state, or lacks efficacy.
 32. The method of claim 30wherein the subject is refractory to therapy with the TNF-α blockingagent.
 33. The method of claim 30 wherein the subject has tuberculosis,an opportunistic infection, or a pathogenic bacterial infection.
 34. Themethod of claim 30 wherein the subject has glomerulonephritis.
 35. Themethod of claim 30 wherein the subject has a demyelinating syndrome. 36.The method of claim 30 wherein the subject has a lupus-like reaction.37. A method comprising: detecting an adverse event in a human subjectwho has rheumatoid arthritis, wherein the subject is being treated witha TNF-α blocking agent, but not a TWEAK blocking agent; andadministering, to the subject, a TWEAK blocking agent in an amount andfor a time effective to provide an overall therapeutic effect.
 38. Themethod of claim 37 further comprising withdrawing the TNF-α blockingagent.
 39. The method of claim 37 wherein the adverse event comprises alupus-like reaction.
 40. The method of claim 37 wherein the adverseevent comprises a bacterial or opportunistic infection.
 41. The methodof claim 37 wherein the adverse event comprises tuberculosis.
 42. Amethod comprising: administering, to a human subject who has rheumatoidarthritis, and who is being or has been withdrawn from a DMARD otherthan a TWEAK blocking agent, a TWEAK blocking agent in an amount and fora time to provide a therapeutic effect.
 43. The method of claim 42wherein the DMARD is methotrexate, parenteral gold, sulphasalazine, orhydroxychloroquinone.
 44. The method of claim 42 wherein the DMARD isother than a TNF-α blocking agent.
 45. The method of claim 42 whereinthe DMARD is withdrawn due to toxicity or to lack of efficacy.
 46. Amethod comprising: administering, to a human subject who has aninflammatory disorder, a TWEAK blocking agent in combination with aTNF-α blocking agent, in amounts and for a time to provide a therapeuticeffect, wherein the inflammatory disorder is one that a TNF-α blockingagent does not exacerbate.
 47. The method of claim 46 wherein theinflammatory disorder is psoriatic arthritis, ankylosing spondylitis,inflammatory bowel disease, psoriasis, or inflammatory myositis.
 48. Themethod of claim 46 wherein the subject is less than 17 years of age, andthe disorder is juvenile rheumatoid arthritis or pediatric psoriasis.49. A method of reducing joint inflammation, the method comprisingidentifying a subject having joint inflammation, and administering tothe subject a TWEAK blocking agent in combination with a TNF-α blockingagent.
 50. The method of claim 49 wherein the subject has rheumatoidarthritis.
 51. The method of claim 49 wherein the TWEAK blocking agentis an antibody that binds to TWEAK.
 52. The method of claim 49 whereinthe TWEAK blocking agent comprises an antibody that binds to a TWEAKreceptor.
 53. The method of claim 49 wherein the TWEAK blocking agentcomprises a soluble form of a TWEAK receptor.
 54. The method of claim 53wherein the soluble form of the TWEAK receptor is fused with an antibodyFc region.